Metalloprotein Active Site Assembly E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Serie: EIC Books
• Sprache: Englisch

Seitenzahl: 1404

Veröffentlichungsjahr: 2017

Beschreibung

Summarizes the essential biosynthetic pathways for assembly of metal cofactor sites in functional metalloproteins

Metalloprotein Active Site Assembly focuses on the processes that have evolved to orchestrate the assembly of metal cofactor sites in functional metalloproteins. It goes beyond the simple incorporation of single metal ions in a protein framework, and includes metal cluster assembly, metal-cofactor biosynthesis and insertion, and metal-based post-translational modifications of the protein environments that are necessary for function. Several examples of each of these areas have now been identified and studied; the current volume provides the current state-of-the-art understanding of the processes involved.

An excellent companion to the earlier book in this series Metals in Cells—which discussed both the positive and negative effects of cellular interactions with metals—this comprehensive book provides a diverse sampling of what is known about metalloprotein active site assembly processes. It covers all major biological transition metal components (Mn, Fe, Co, Ni, Mo), as well as the other inorganic components, metal-binding organic cofactors (e.g., heme, siroheme, cobalamin, molybdopterin), and post-translationally modified metal binding sites that make up the patchwork of evolved biological catalytic sites. The book compares and contrasts the biosynthetic assembly of active sites involving all biological metals. This has never been done before since it is a relatively new, fast-developing area of research.

Metalloprotein Active Site Assembly is an ideal text for practitioners of inorganic biochemistry who are studying the biosynthetic pathways and gene clusters involved in active site assembly, and for inorganic chemists who want to apply the concepts learned to potential synthetic pathways to active site mimics.

Leseprobe

Table of Contents

Cover

Title Page

Copyright

Encyclopedia of Inorganic and Bioinorganic Chemistry

Contributors

Series Preface

Volume Preface

Periodic Table of the Elements

Part 1: Assembly and Trafficking of Simple Fe-S Clusters

Nif System for Simple [Fe–S] Cluster Assembly in Nitrogen-Fixing Bacteria

1 Introduction

2 The Nif System as a Model for Analysis of Simple [Fe–S] Cluster Assembly

3 Analysis of the Mo-Dependent Nitrogen-Fixing System in

A. vinelandii

4 Genetic Phenotypes and Biochemical Features Indicated a Role for NifU and NifS in [Fe–S] Cluster Formation

5 NifS Cysteine Desulfurase

6 NifU Provides a Scaffold for [Fe–S] Cluster Assembly

7 NifU and NifS as the Minimum Set for the Assembly and Transfer of Fe–S Clusters

8 The NifS/NifU [Fe–S] Cluster Assembly Toolkit Provides a Paradigm for Simple [Fe–S] Cluster Assembly

9 Functional Cross Talk Between [Fe–S] Cluster Biosynthetic Systems

10 Concluding Remarks

11 Acknowledgments

12 Related Articles

13 Abbreviations and Acronyms

14 References

Iron–Sulfur Cluster Assembly in Bacteria and Eukarya using the ISC Biosynthesis Machinery

1 Introduction

2 Core Fe–S Cluster Assembly Step

3 Fe–S Cluster Transfer Step

4 From the Mitochondria to the Cytosol

5 Unsolved Questions

6 Related Articles

7 Abbreviations and Acronyms

8 References

The Suf System in Archaea, Bacteria, and Eukaryotic Organelles

1 Introduction

2 Bacterial Suf Pathways: Diverse Roles in Fe–S Cluster Biogenesis

3 Archaea and the Origin of the Suf Pathway

4 Eukaryotic Suf Systems: A Role for Suf in Plastid Organelles and in the Cytoplasm

5 Conclusions

6 Related Articles

7 Abbreviations and Acronyms

8 References

Roles of Class II Glutaredoxins in the Maturation of Fe–S Proteins

1 Introduction

2 The Cellular Maturation of Fe–S Proteins in Model Organisms

3 Involvement of Glutaredoxins in the Maturation of Fe–S Proteins in Eukaryotes

4 Spectroscopic and Structural Data for Fe–S Bridged Complexes Involving Class II GRXs

5 Molecular Interactions Between Class II GRXs and their Partners

6 Conclusions

7 Related Articles

8 Abbreviations and Acronyms

9 References

Part 2: Assembly of Complex and Heterometallic Fe-S Cluster Active Sites

Nitrogenase Metallocluster Assembly

1 Introduction

2 Properties of NifDK Metalloclusters

3 Biosynthetic Factors

4 M-Cluster Biosynthesis

5 Biosynthesis of P Cluster

6 Discussion and Future Directions

7 Acknowledgments

8 Related Articles

9 Abbreviations and Acronyms

10 References

Metallocluster Assembly: Maturation of [FeFe]-Hydrogenases

1 Introduction

2 The Metal Center of [FeFe]-Hydrogenases

3 A Protein Machinery for [FeFe]-Hydrogenase Maturation

4 The Hyd Proteins

5 Mechanism of Maturation of [FeFe]-Hydrogenases

6 Maturase-Free Chemical Maturation: A Unique Technological Tool

7 Conclusions

8 Acknowledgments

9 Abbreviations and Acronyms

10 References

CO Dehydrogenase and Acetyl-CoA Synthase

1 Introduction

2 Maturation of Ni,Fe-CODHs

3 Maturation of Ni,Fe-Containing ACS

4 Outlook

5 Acknowledgments

6 Related Articles

7 Abbreviations and Acronyms

8 References

Part 3: Assembly of Homometallic and Heterometalic Cu Cluster Active Sites

Assembly of Dinuclear Copper Center in Tyrosinases and Hemocyanins

1 Introduction

2 Tyrosinase and Hemocyanin: An Overview

3 Bacterial Tyrosinase

4 Mammalian Tyrosinase

5 Fungal Tyrosinase

6 Plant Tyrosinase (Catechol Oxidase) and Molluskan and Arthropod Hemocyanin

7 Summary

8 Related Articles

9 Abbreviations and Acronyms

10 References

Multicopper Oxidases

1 Introduction

2 Overview of MCO Structure andCopper-Coordination Sites

3 MCOs In Vitro: Interconversion of Apo and Holo Forms and Their Stability

4 Copper Redox State as a Probe of Copper-Site Assembly: Fet3

5 The

Bacillus subtilis

CotA MCO: Tracking the Protein and Metal in vivo and in vitro

6 Cellular Trafficking of MCO Proteins andProsthetic Group Copper

7 Summary

8 Related Articles

9 Abbreviations and Acronyms

10 References

Assembly of the Redox-Active Metal Centers of Cytochrome

c

Oxidase

1 Introduction

2 Heme a Biosynthesis and Insertion

3 Assembly of the Cu

B

Center

4 Assembly of the Cu

A

Center

5 Metallation of Copper Chaperones Involved in C

c

O Assembly

6 Acknowledgments

7 Abbreviations and Acronyms

8 References

CuA and CuZ Center Assembly in Nitrous Oxide Reductase

1 Introduction

2 The Enzyme Nitrous Oxide Reductase

3 Assembly of Copper Centers into Nitrous Oxide Reductase

4 Conclusions

5 Acknowledgments

6 Related Articles

7 Abbreviations and Acronyms

8 References

MoCu CO Dehydrogenase and its Active-Site Assembly

1 Introduction

2 Chemolithoautotrophic Growth with CO

3 MoCu CO Dehydrogenase of

Oligotropha carboxidovorans

4 CO Dehydrogenase Active-Site Assembly

5 Function of CoxG as Membrane Anchor

6 Summary

7 Related Articles

8 Abbreviations and Acronyms

9 References

10 Further Reading

Part 4: Assembly of Homometallic and Heterometallic Mn Clusters

Homo- and Heterometallic Dinuclear Manganese Proteins: Active Site Assembly

1 Introduction

2 Homometallic Manganese Proteins and their Cofactor Assembly

3 Heterometallic Manganese–Iron Proteins and their Cofactor Assembly

4 Conclusions

5 Acknowledgments

6 Related Articles

7 Abbreviations and Acronyms

8 References

Biogenesis and Assembly of the CaMn

4

O

5

Core of Photosynthetic Water Oxidases and Inorganic Mutants

1 Introduction: Brief History, Biogeological Impact

2 Structure and Evolution of PSII

3 WOC Operation

4 Manganese Speciation and Redox EquilibriaRelevant to Cells

5 In Vitro Photoassembly

6 In Vivo Photoassembly

7 Future Directions

8 Acknowledgments

9 Related Articles

10 Abbreviations and Acronyms

11 References

12 Further Reading

Part 5: Assembly of Homometallic and Heterometallic Ni Clusters

Urease Activation

1 Introduction

2 Accessory Proteins Required for Urease Active Site Assembly

3 Acknowledgments

4 Related Articles

5 Abbreviations and Acronyms

6 References

Insights into [NiFe]-Hydrogenase Active Site Metallocluster Assembly

1 Introduction

2 The Accessory Protein Machinery

3 Cofactor Biosynthesis

4 Nickel Insertion

5 The Conformational Switch and Active Site Closure

6 Protein Assembly

7 Perspectives

8 Acknowledgments

9 Related Articles

10 Abbreviations and Acronyms

11 References

Part 6: Assembly of Cofactors for Binding Active-site Metal Centers

Moco in Mo/W Enzymes

1 Introduction

2 The Biosynthesis of the Molybdenum Cofactors

3 The Biosynthesis of the Molybdenum Cofactors in Humans

4 Conclusions

5 Acknowledgments

6 Related Articles

7 Abbreviations and Acronyms

8 References

Heme Biosynthesis

1 Introduction

2 Synthesis of Aminolevulinic Acid (ALA)

3 Conversion of ALA to Uroporphyrinogen III

4 Siroheme-Dependent (SHD) Pathway

5 Further Preparation for the Coproporphyrin and Protoporphyrin-Dependent Pathways

6 Coproporphyrin-Dependent (CPD) Pathway

7 Protoporphyrin-Dependent (PPD) Pathway

8 Parasite Heme Biosynthesis

9 Regulation of Heme Synthesis

10 Future Directions in Heme Synthesis

11 Acknowledgments

11 Related Articles

13 Abbreviations and Acronyms

14 References

Siroheme Assembly and Insertion to Nitrite and Sulfite Reductase

1 Siroheme Function

2 Siroheme Biogenesis: CysG

3 Cofactor Assembly

4 Related Articles

5 Abbreviations and Acronyms

6 References

Biosynthesis of Coenzyme F430 and the Posttranslational Modification of the Active Site Region of Methyl-Coenzyme M Reductase

1 Introduction

2 Coenzyme F430 Biosynthesis

3 MCR Posttranslational Modifications

4 MCR Catalysis in ANME

5 Conclusions

6 Related Articles

7 Abbreviations and Acronyms

8 References

Coenzyme B

12

Biosynthesis in Bacteria and Archaea

1 Introduction

2

De Novo

Corrin Ring Biosynthesis

3 The Nucleotide Loop Assembly (NLA) Pathway

4 Salvaging Complete and Incomplete Corrinoids

5 Biosynthesis of DMB and other Benzimidazoles

6 Concluding Remarks

7 Acknowledgments

8 Glossary

9 Abbreviations and Acronyms

10 References

Crosslinked Cys–Tyr Free Radical Redox Cofactor

1 Introduction

2 Cys–Tyr Copper Metalloradical Active Site

3 Cofactor Self-Processing Reaction

4 Conclusions

5 Acknowledgments

6 Related Articles

7 Abbreviations and Acronyms

8 References

Topaquinone Biogenesis and Lysyl Tyrosine Quinone Biogenesis in Cu Amine Oxidases

1 Introduction

2 Structural Background and Key Insights

3 Mechanistic Studies and the Characterization of Intermediates in TPQ Biogenesis

4 The Biogenesis of LTQ

5 Inorganic Reactivity Considerations

6 Acknowledgments

7 Related Articles

8 Abbreviations and Acronyms

9 References

Index

Abbreviations and Acronyms used in this Volume

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Guide

cover

Table of Contents

Begin Reading

List of Illustrations

Nif System for Simple [Fe–S] Cluster Assembly in Nitrogen-Fixing Bacteria

Figure 1 [Fe–S] clusters associated with nitrogen-fixing proteins. Atoms are color-coded in green (iron), yellow (sulfur), magenta (molybdenum), red (oxygen), and gray (carbon)

Figure 2 Nitrogen fixation-associated gene regions in the

Azotobacter vinelandii

genome. Mo-dependent (

nif

), V-dependent (

vnf

), and iron-only (

anf

) nitrogen fixation systems are shown. Genes encoding nitrogenase catalytic components are shown in green, regulatory proteins are shown in blue, and genes essential to all three nitrogenase systems are indicated by red borders. Coding sequences having paralogs also located elsewhere in the genome, but whose expression is not controlled by

nif

regulatory elements, are shown in gray. Various

nif

and

nif

-associated genes encoding proteins having [Fe–S] cluster-binding motifs are indicated by a dot below the designated gene. Arrows above the gene segments indicate the location of known or proposed promoters controlled by nitrogen fixation–specific regulatory elements

Figure 3

A. vinelandii

nitrogenase structures. Ribbon diagram of MoFe protein and Fe protein complex in the presence of Mg-ADP and the ATP analog, Mg-AMPPCP (PDB ID 4WZA). (a) A catalytic unit comprised of one Fe protein dimer showing each subunit in blue and cyan and one

α

/

β

dimer shown in dark and light pink, respectively. (b) The entire complex with two catalytic units. [Fe–S] clusters, Mg-ADP, and Mg-AMPPCP are shown in space filling models

Figure 4 Two-dimensional polyacrylamide gel electrophoresis profile of soluble

A. vinelandii

extracts prepared from cells cultured under nitrogen-fixing conditions. The gel illustrates the high level of accumulation of Fe protein (H) and MoFe protein (D and K for

α

and

β

subunits, respectively) in nitrogen-fixing cells

Figure 5 Proposed catalytic mechanism NifS cysteine desulfurase reaction. Under resting state, NifS Lys

202

is covalently attached via a Schiff base (internal Lys-aldimine,

1

). The binding of Cys results in the formation of the external Cys-aldimine intermediate (

2

). The abstraction of the substrate

α

-proton then leads to a rearrangement to form the Cys-quinonoid (

3

) and Cys-ketimine (

4

) intermediates. The active site Cys

325

, then, promotes the nucleophilic attack onto the thiol group of the substrate, leading to the formation of the persulfide enzyme and Ala-enamine intermediates (

5

). The reaction is completed through sequential formation of Ala-ketimine (

6

), Ala-aldimine (

7

), and restoration of the Lys-aldimine bond (

1

). The second half of the NifS cysteine desulfurase mechanism is the sulfur transfer reaction from the persulfide intermediate to NifU during Fe–S cluster assembly

Figure 6 NifU is an [Fe–S] cluster assembly scaffold containing three functional domains. The critical cysteine residues located in each domain are indicated. The N-terminal IscU-type domain (purple) is able to accommodate transient [2Fe–2S] and [Fe–4S] clusters, while the C-terminal Nfu-type (cyan) is able to accommodate transient [4Fe–4S] clusters. The central domain (gray) contains a stable ferredoxin-like [2Fe–2S] cluster. The working model involves the sequential assembly of [2Fe–2S] clusters, followed by reductive coupling to form a [4Fe–4S] cluster within the N-terminal domains and transfer to the C-terminal domain

Figure 7 The [4Fe–4S] cluster of Fe protein can be removed by incubation with chelator agents. Upon the binding of ATP, Fe protein undergoes a conformational change that exposes its [4Fe–4S] cluster, which can be removed by dipyridyl to generate the apo form of the enzyme

Figure 8 Basic steps in [Fe–S] cluster assembly. NifS (yellow) is a PLP-dependent cysteine desulfurase that catalyzes the Cys:NifU sulfur transfer reaction through formation of alanine. NifU (gray) serves as the scaffold for the synthesis and transfer of a [4Fe–4S] cluster for the activation of Fe protein (blue)

Figure 9 [Fe–S] cluster biosynthetic gene regions in

A. vinelandii

. Genes encoding paralogs are color-coded accordingly.

iscS

and

nifS

are cysteine desulfurases, highlighted in yellow. They are respectively responsible for sulfur activation and transfer to the [Fe–S] cluster assembly scaffold sites provided within

iscU

or the N-terminal domain of

nifU

, highlighted in lilac. Cysteine biosynthesis is boosted by the activity of an

O

-acetyl serine transferase encoded by

cysE

, highlighted in brown. The transfer of [Fe–S] clusters is respectively assisted by

nfuA

or the C-terminal domain of

nifU

, highlighted by cyan. The A-type proposed intermediate [Fe–S] cluster carriers designated

iscA

nif

,

iscA

, and

erpA

are highlighted in pink. Chaperones associated with the Isc system are designated

hscA

and

hscB

and are highlighted in green. Electron transfer reactions that are proposed to occur during [Fe–S] cluster synthesis could involve a ferredoxin, highlighted in green, or the central domain of

nifU

, highlighted in gray

Iron–Sulfur Cluster Assembly in Bacteria and Eukarya using the ISC Biosynthesis Machinery

Figure 1 Iron–sulfur cluster assembly process. Two main steps: the core assembly step (1) and the Fe–S cluster transfer step (2)

Figure 2 Schematic model for Fe

2

S

2

and Fe

4

S

4

clusters assembly on a scaffold protein. CD, cysteine desulfurase

Figure 3 Proposed mechanism for Fe

2

S

2

cluster assembly on the scaffold protein. The bacterial system is taken as an example

Figure 4 Fe–S cluster transfer in procaryotes (a) and in eukaryotes (b). The Fe–S transfer from the scaffold can be direct or indirect through Fe–S carrier or protein factors

The Suf System in Archaea, Bacteria, and Eukaryotic Organelles

Figure 1 Structure of the

E

.

coli

succinate dehydrogenase complex with Fe–S clusters highlighted as spheres in the structure with ball and stick representation to the right. Ubiquinone (UQ), flavin (FAD), heme

b

, and Fe–S cluster side chain ligands are shown as sticks

Figure 2 Proposed model for Suf function

Figure 3 Alignment of CsdE (from its co-structure with CsdA) with resting SufE. Active site Cys residues SufE C51 and CsdE C61 are shown

Figure 4 (a) Overall structure of SufBC

2

D with Hg

2+

shown as gray spheres (from PDB 5AWG). (b) Zoom of the metal-binding site at the SufB–SufD interface with residues (sticks) labeled

Figure 5 Proposed model for the housekeeping Suf pathway in Gram-positive bacteria. Gray arrows indicate putative steps in Fe–S cluster biogenesis that have not been shown experimentally

Figure 6 The Suf system in plastids. Arrows indicate likely or hypothesized pathways in the biogenesis of Fe–S clusters. SufS and SufE1 form a cysteine desulfurase and SufBCD likely forms the scaffold. The direct source of Fe remains unclear. Four potential downstream carriers exist (GRX, SufA, NFU, and HCF101). See text for details

Roles of Class II Glutaredoxins in the Maturation of Fe–S Proteins

Figure 1

Basic principles for Fe–S cluster biosynthesis and Fe–S protein maturation.

The biogenesis of Fe–S clusters can be divided into two principal steps: (i) the

de novo

Fe–S cluster synthesis on a scaffold protein using sulfur atoms provided by a cysteine desulfurase in the form of a persulfide group and iron atoms from unknown origin, (ii) the transfer to recipient apoproteins through the action of transfer proteins that transiently bind the cluster in association with targeting factors that are thought to recruit the appropriate target proteins

Figure 2

Models for the Fe–S cluster assembly machineries in eukaryote cells.

Recognized or putative molecular components participating in the chloroplastic SUF (a), the mitochondrial ISC (b), and the cytosolic CIA (c) machineries. The yeast protein nomenclature is used for the ISC and CIA machineries and the

Arabidopsis

nomenclature for the SUF machinery. In violet are proteins associated with the cysteine desulfuration step, in red the proteins associated with electron donation, in green the scaffold proteins, in blue the Fe–S cluster transfer proteins and targeting factors, and in orange the final acceptors

Figure 3

GSH and [Fe

2

S

2

] cluster binding sites in class II GRXs.

(a) The GSH molecule (colored in red) is stabilized by a disulfide bridge with the catalytic cysteine present at the N-terminal extremity of

α

2 (colored in purple) and through hydrogen bonds with a Lys found in the loop between

β

1 and

α

2 (colored in yellow), a Lys and an Arg present in

α

3 (colored in orange), the Val of the TVP motif (colored in blue), and residues following the GG motif (colored in green). Hydrogen bonds are represented as white dashed lines and the disulfide bond between the GSH molecule and the catalytic cysteine as a black dashed line. (b) The [Fe

2

S

2

] cluster is located at the interface of a GRX homodimer. The cluster is ligated by the catalytic cysteine of both monomers and by two GSH molecules. The [Fe

2

S

2

] cluster, catalytic cysteines, and GSH molecules are shown as sticks

Figure 4

Structural alignment of BOLA and GRX sequences highlighting known interacting residues. (

A) Multiple sequence alignment of

Arabidopsis thaliana

BOLA2 and

Homo sapiens

BOLA1, BOLA2, and BOLA3 generated using CLUSTALO. The consensus secondary structure elements are indicated as red cylinders (helices) and yellow arrows (

β

-strands). The two invariant residues of the BOLA family are indicated by stars. The conserved histidine and cysteine residues of BOLA_H and BOLA_C members, respectively, are highlighted on a green background. Residues involved in BOLA–GRX interactions were determined by NMR spectroscopy. Those showing significant chemical shift perturbation upon

15

N-BolA–Grx apo-heterodimer formation (a) are displayed in red. Additional residues found upon

15

N-BolA–Grx holo-heterodimer formation (h) are displayed in blue.

15, 41, 47

Mutations found in human patients affected for BOLA3 are also pointed. (B) Multiple sequence alignment of

Arabidopsis thaliana

GRXS14 and

Homo sapiens

GRX3A, GRX3B (A and B represent the first and the second GRX domain of GRX3) and GRX5. Residues involved in GRX–BOLA and HsGRX5–ISCA1 interactions were determined by NMR spectroscopy. Those showing significant chemical shift perturbation upon

15

N-GRX–BOLA apo-heterodimer formation (a) are displayed in red. Additional residues found upon

15

N-GRX–BOLA holo-heterodimer formation (h) are displayed in blue.

15, 41, 47

Residues found upon

15

N-HsGRX5–ISCA1 formation are displayed in red.

51

Mutations found in the glrx5 gene of human patients are also pointed out

Figure 5

Mapping of BOLA–GRX interfacing residues

. The interactions between BOLA and GRX couples from various organisms were studied by NMR spectroscopy.

15, 41, 47

All residues showing significant chemical shift perturbation upon apo-heterodimer formation (HsBOLA1–GRX5, HsBOLA3–GRX5, HsBOLA2–GRX3, and AtBOLA2–GRXS14) are merged, displayed in red and represented for BOLAs (a) and GRXs (b). All residues showing significant chemical shift perturbation upon holo-heterodimer formation (HsBOLA1–GRX5–[Fe

2

S

2

] and HsBOLA3–GRX5–[Fe

2

S

2

]) are merged and represented for BOLAs (c) and GRXs (d). Additional residues found upon holo-heterodimer formation are colored in blue. The invariant histidine of BOLAs and the Fe–S ligating cysteine of GRXs are shown in sticks. The [H/C]-loop containing the conserved histidine or cysteine residue in the BOLA_H or BOLA_C isoforms is circled

Nitrogenase Metallocluster Assembly

Figure 1 Molecular structure of

A. vinelandii

Mo-nitrogenase NifDK/NifH complex stabilized by ADP · AlF

4

−

with the proteins and

α

2

β

2

-subunits labeled (a) and representation of the catalytic complex with metalloclusters and nucleotides emphasized (b) to highlight the flow of electrons during substrate reduction. Atoms are represented by spheres colored as follows: Fe, orange; S, yellow; Mo, cyan; C, light gray; N, blue; O, red; Mg, green; Al, dark gray; F, light blue. Images were generated with PYMOL (PBD ID: 1N2C)

Figure 2 Molecular structures of the M cluster (a) and the P

N

(b) and P

OX

(c) states of the P cluster in NifDK, with cluster atoms drawn as spheres and amino acid side chain ligands drawn as sticks. Atoms are colored as follows: Fe, orange; S, yellow; Mo, cyan; C, light gray; N, blue; O, red. Images were generated with PYMOL (PBD IDs: 1M1N, 3MIN)

Figure 3 The versatile reductive actions of NifH: maturation of L cluster to M cluster on the scaffold NifEN through the insertion of Mo and homocitrate (a); reductive coupling to mature the P* cluster to P cluster on NifDK (b); and as the reductase association partner for NifDK during catalytic substrate reduction (c). All three processes appear to be dependent on MgATP hydrolysis and require prior reduction of NifH by a suitable exogenous electron source

Figure 4 Schematic outlining M-cluster biosynthesis. The mobilization of Fe and S through the actions of NifS and NifU generates [Fe

4

S

4

] clusters that are delivered to NifB. On NifB, a pair of [Fe

4

S

4

] clusters termed the K cluster is coupled to form the [Fe

8

S

9

C]-core L cluster in a SAM-dependent reaction. After transfer to the scaffold protein NifEN, the L cluster is matured to the [MoFe

7

S

9

C]-core M cluster through concomitant Mo and homocitrate insertion from NifH. Finally, the M cluster is delivered to the catalytic Mo-nitrogenase component NifDK. Drawn cubes represent [Fe

4

S

4

] clusters, and the K, L, and M clusters are drawn as appropriately labeled lozenges. Solid arrows represent intraprotein molecular transformations, while dashed arrows indicate interprotein molecular transfers

Figure 5 Proposed mechanism of SAM-mediated maturation of K cluster to L cluster on NifB. A first equivalent of SAM methylates a sulfur atom on the K cluster, producing

S

-adenosyl homocysteine (SAH). A second equivalent of SAM is reduced by the SAM-responsive [Fe

4

S

4

] cluster on NifB to homolytically generate a 5′-deoxyadenosyl (5′-dA

⋅

) radical species and methionine (MetH). A hydrogen atom of the

S

-CH

3

group is removed by 5′-dA

⋅

to yield a K-cluster-bound

S

-CH

2

⋅

species and 5′-dAH. Following introduction of a “ninth” sulfur atom, loss of two protons, and bond rearrangements, the L cluster is formed

Figure 6 Reactivity of

S

-adenosylmethionine (SAM) with NifB variants heterologously expressed from

E. coli

. Perpendicular-mode EPR spectra of NifB variants of dithionite-reduced (a) and IDS-oxidized (b) samples before (black line) and after (gray line) treatment with SAM. Spectra are for NifB from

M. acetivorans

(

Ma

NifB, a,b top) and

M. thermoautotrophicus

(

Mt

NifB, a,b bottom), with indicated

g

values. HPLC traces from SAM and cleavage product standards (c, top) and after incubation of

Ma

NifB (c, middle) or

Mt

NifB (c, bottom) with SAM and dithionite. (1) His-tagged enzymes

Ma

NifB (D1) or

Mt

NifB (E1) bound to Ni-affinity (IMAC) resin after incubation with [methyl-

14

C] SAM. (2) Following incubation with [methyl-

14

C] SAM and nontagged

A. vinelandii

apo-NifEN, NifH, and apo-NifDK, minimal radiolabel remains on IMAC resin for

Ma

NifB (D2) and

Mt

NifB (E2). (3) Reconstituted nontagged

A. vinelandii

NifDK (

Av

NifDK*) bound to anion-exchange (DEAE) resin after incubation with [methyl-

14

C] SAM and His-tagged enzymes apo-NifEN, NifH, and

Ma

NifB (D3) or

Mt

NifB (E3). (4) L clusters extracted from IMAC resin (D1, E1) and M clusters extracted from DEAE resin (D3, E3). Also included in assays (2) and (3): dithionite, MgATP, MoO

4

2−

, and homocitrate. BG, background

Figure 7 Maturation of L cluster to M cluster on NifEN (NifEN

M

, a) and direct transfer of the M cluster to cofactor-deficient NifDK (apo-NifDK, b). First, NifEN receives L cluster from the scaffold protein NifB (a). Through the actions of the MgATP-dependent Mo/homocitrate insertase NifH, surface-exposed L cluster is matured to M cluster, followed by a conformation change that buries the cofactor within NifEN. The M cluster is then delivered to a higher-affinity cofactor-binding site on apo-NifDK directly through protein–protein interactions (b). Receipt of M cluster triggers a conformational change in NifDK that locks in the cofactor to form the holoenzyme

Figure 8 Comparison of M cluster on NifDK to M cluster matured from L cluster on NifEN (NifEN

M

). Fe K-edge XAS spectra (a) of M cluster extracted into NMF from either NifEN

M

(gray) or NifDK (black). Mo K-edge XAS spectra (b) of M cluster extracted into NMF from either NifEN

M

(gray) or NifDK (black). Perpendicular-mode EPR spectra (c) of dithionite-reduced NifEN

M

(gray) and NifDK (black), with indicated

g

values. Measured Fe amounts (d) determined either by ICP-OES metals analysis (gray bars, total mol Fe contained/mol protein) or chelation studies with bathophenanthroline disulfonate (black diagonal-lined bars, total mol Fe chelated/mol protein). Iron concentrations are shown for L-cluster-deficient NifEN (apo-NifEN), NifEN as isolated (NifEN

L

), and NifEN after incubation with NifH, dithionite, MgATP, MoO

4

2−

, and homocitrate (NifEN

M

)

Figure 9 Electrostatic potential representations showing the

α

-subunits of cofactor-deficient NifDK (apo-NifDK), NifEN with bound L cluster, and M-cluster-containing NifDK (holo-NifDK). An illustrated cross-section of apo-NifDK highlights amino acid side chains associated with the M-cluster insertion path. First, NifEN delivers mature M cluster to apo-NifDK, where the cluster is believed to interact with the positively charged “lid loop” region (A), in particular with the ligand His

α

362

. The M cluster diffuses further into apo-NifDK by putative interaction with the cluster-binding “His triad” (B), consisting of His

α

274

, His

α

451

, and His

α

442

. At this point, the M cluster is in a similar position to L cluster as observed on NifEN. Finally, the “switch/lock” region (C) His

α

442

residue binds to M cluster, and Trp

α

444

rotates to lock in the cofactor. This closed conformation is evident with the holo-NifDK structure, where the M cluster is completely embedded and obscured within the protein matrix. Images were generated with PYMOL (PBD IDs: 1L5H, 3PDI, 1M1N) with isopotential surfaces calculated using Adaptive Poisson–Boltzmann Solver (APBS, www.poissonboltzmann.org)

Figure 10 P-cluster biosynthesis on NifDK (a) and associated EPR spectra (b,c). The actions of NifS and NifU establish the P* cluster, a pair of [Fe

4

S

4

]-like clusters, on each

αβ

-subunit half of NifDK. The “first” P* cluster requires reductive coupling with NifH to form P cluster, while coupling of the “second” P* cluster requires the actions of both NifZ and NifH (a). The variant designation labels indicate isolable deletion mutants that possess the illustrated cluster composition:

ΔnifH

NifDK, two P* clusters;

ΔnifBΔnifZ

NifDK, one each of P* and P clusters; and

ΔnifB

NifDK, two P clusters. Dithionite-reduced (b) and IDS-oxidized (c) EPR spectra of cluster species on

ΔnifH

NifDK before (black line) and after (gray line) maturation with NifH, MgATP, and dithionite. Maturation of

ΔnifH

NifDK causes loss of the

S

= 1/2 perpendicular-mode EPR signal (b) from P* cluster and concomitant increase of the parallel-mode

g

= 11.8 feature (c) that is characteristic of P cluster

Metallocluster Assembly: Maturation of [FeFe]-Hydrogenases

Figure 1 Schematic representation of the active sites of [FeFe]-, [NiFe]-, and [Fe]-hydrogenase

Figure 2 Chemical reconstitution of HydA [2Fe] subcluster. HydF loaded with synthetic diiron complexes mimicking the [2Fe] subcluster (complexes

1

,

2

, and

3

) can transfer the diiron unit to HydA

Figure 3 FTIR spectra of HydF. CaHydF: HydF isolated from

C. acetobutylicum

host expressing all the clostridial maturases (HydEFG).

31

x

-HydF (

x

=

1

or

2

): chemically reconstituted HydF from

Thermosipho melanesiensis

with synthetic complexes

1

and

2

.

33

Protein samples have been treated with sodium dithionite (NaDT)

Figure 4 (a) Structure of the dimer of TmeHydF (pdb 5kh0). The GTPase, dimerization, and cluster-binding domains of one subunit are colored in orange, pink, and blue respectively. Helices, strands, and loops of the second subunit are colored in green, yellow, and white respectively. The iron–sulfur clusters of the two subunits are displayed as spheres with the sulfur in yellow and the iron in red. Residues in sticks correspond to C298, C349, C352, and E305. (b) Close-up of the iron–sulfur cluster-binding site. (c) Surface representation of the region around the active site showing the cavity near the iron–sulfur cluster displayed as spheres. Color scheme as in (a). The white surface corresponds to the linker between the GTPase and dimerization domains

Figure 5 Structure of TmHydE (pdb 3cix). The [Fe–S] clusters are shown as spheres with sulfur colored in yellow and iron in red, SAM is displayed as sticks. HydE contains one [4Fe–4S] cluster associated with radical SAM activity ([4Fe–4S]

RS

). A second cluster is shown ([Fe–S]

AUX

) but it is of variable constitution and can be removed without affecting the enzyme activity. The substrate-binding site cavity is shown as transparent purple surface

Figure 6 Structure of TiHydG. (a) Overall structure of TiHydG (pdb 4wcx). The two iron–sulfur clusters are depicted as spheres with sulfur and iron colored in yellow and red, respectively. SAM is represented as sticks. The substrate-binding site cavity linking the two clusters is shown as transparent purple surface. (b) [4Fe–4S] cluster associated with radical SAM where tyrosine is converted to CO and CN

−

(c) Auxiliary [Fe–S] in its [5Fe–5S] form. The fifth iron (in red) is coordinated by His265, two water molecules (magenta), a sulfide, and an unidentified amino acid (in sticks with carbons and bonds in black, nitrogen and oxygen in blue and light red, respectively)

Figure 7 Proposed mechanism of HydG. Tyrosine is fragmented into

p

-cresol and dehydroglycine (DHG). DHG is then converted into CO (in red) and CN

−

(in blue), which are coordinated by the “dangling Fe” of the [5Fe–4S]

AUX

cluster. Two molecules of DHG (DHG

1

and DHG

2

) are needed to provide the final product (complex B) with a Fe(CO)

2

(CN) unit

Figure 8 [FeFe]-hydrogenase (HydA) maturation process: assembly of the [2Fe] subcluster. HydG and HydE produce the [(

κ

3

-Cys)Fe(CO)

2

(CN)]

−

and the adt

2−

synthons, respectively. HydF captures these synthons, assembles a diiron precursor (presumably

1

), and transfers it to HydA where it converts into the catalytically active [2Fe] subcluster

Figure 9 A common theme in HydA maturation: a [4Fe–4S] cluster controls organometallic species in HydG, HydF, and HydA

Figure 10 Completely chemical assembly of metallocofactors in HydA from

Megasphaera elsdenii

. The chemical maturation involves two steps: first, all [4Fe–4S] clusters are reconstituted using Fe

2+

and S

2−

(Na

2

S or

l

-Cys in the presence of cysteine desulfurase), and second, the H cluster is completed by adding the synthetic complex

1

CO Dehydrogenase and Acetyl-CoA Synthase

Figure 1 Structure of Ni,Fe-containing carbon monoxide dehydrogenase. (a) Overall structure of CODH2

Ch

in cartoon display, with the two monomers in different colors and the metal clusters shown as spheres with nickel (green), sulfur (yellow), and iron (orange). B′ and C′ denote metal clusters from the same monomer. (b) Metal centers of CODH with same color code as in (a). (c) Ball–stick display of the C cluster. Color code for atoms as in (a) and oxygen in red. (d) Scheme of the C cluster. All protein structure presentations were prepared using PyMol

40

Figure 2 Sequence similarity network of CooC proteins (InterPro database).

62

The network was generated from an all-by-all distance matrix storing Kimura distances calculated from a multiple amino acid sequence alignment. Gray colored lines connect nodes (amino acid sequences) with a Kimura distance below 1.2. Nodes are arranged using the yFiles organic layout of Cytoscape 2.8.3.

63

Large yellow nodes are representative sequences from the genomes of

Carboxydothermus hydrogenoformans

(CooC1

Ch

, CooC2

Ch

, CooC3

Ch

),

Moorella thermoacetica

(CooC

Mt

, AcsF

Mt

), and

Rhodospirillum rubrum

(CooC

Rr

), while red, green, and blue denote the three subgroups

Figure 3 Structure of dimeric CooC1

Ch

in cartoon display. Monomers are distinguished by color (green and cyan) and labels indicate ADP and Zn. The metal-binding site (MBS) and the CAP loop, shielding the MBS from the top, are both labeled. The gray oval indicates the flexible region, which is specific for CooC proteins and changes its structure upon metal binding

Figure 4 Structure of Ni,Fe-containing acetyl-CoA synthase. (a) Overall structure of monomeric ACS

Ch

in cartoon display, with the three domains labeled and the A cluster shown as spheres with nickel (green), sulfur (yellow), and iron (orange). (b) Cartoon display of bifunctional ACS/CODH

Mt

; atoms are shown with same color code as in (a). ACS

open

and ACS

closed

denote the open and closed conformations of the three ACS domains. (c) Ball–stick display of the A cluster. Color code for atoms as in (a). (d) Scheme of the A cluster

Assembly of Dinuclear Copper Center in Tyrosinases and Hemocyanins

Figure 1 Crystal structure of the met-form of sweet potato catechol oxidase from

Ipomoea batatas

(PDB entry: 1BT1). All Figure were produced with the program PyMOL (http://www.pymol.org)

Scheme 1 Three states of dinuclear copper center of TY in the catalytic cycle

Figure 2 Overall structure of

Streptomyces castaneoglobisporus

tyrosinase complexed with the caddie protein. The ribbon view of the copper-binding subunit (tyrosinase, marine) and ORF378 (caddie, pink)

Figure 3 Copper atoms found in the crystal structure of the complex between tyrosinase and Y98F caddie determined based on an anomalous difference Fourier electron density map (PDB entry: 3AX0). Residues of the caddie His82, Met84, and His97 and the TY His54 take two conformers

Figure 4 TY maturation and trafficking through the secretory pathway. TY is translated and folded in the endoplasmic reticulum (ER). The export-competent tyrosinase is transported to the

cis

-Golgi network. In the

trans

-Golgi network (TGN), the copper is loaded and is delivered to the melanosomes, where TY gains the copper again to compensate for the insufficient copper

Figure 5 Crystal structure of pro-tyrosinase, tyrosinase-encoding gene (

melB

) product from

Aspergillus oryzae

(AoTY) (PDB code: 3W6W). Copper ions are shown in green. The two subunits with the two domains are shown in cartoon.

Figure 6 Comparison of AoTY (marine, PDB code: 3W6W) with AbTY4 (salmon, PDB code: 4OUA). Copper site is shown as green spheres. Superposition of monomers of AoTY and AbTY (salmon, PDB code: 4OUA). Copper grapplers of AoTY and AbTY are shown in magenta and marine. The broken line indicates disordered loop region

Scheme 2 Schematic representation of copper incorporation in tyrosinase

Figure 7 The structure of the functional unit

d

of molluskan HC (PDB code: 4YD9). Highly conserved sulfur-containing residues surrounding the dinuclear copper center (yellow sticks)

Multicopper Oxidases

Figure 1

The T1 Cu ligand loop

. These structures illustrate the

β

-turn that connects the anti-parallel strands in the cupredoxin fold that provide three of the four ligands common to the T1 Cu site in small “blue” copper proteins and MCOs (multicopper oxidases). The fourth ligand is contributed by a histidine ∼60 residues N-terminal of this motif: H85 (rusticyanin); H413 (Fet3); and H994 (hCp). Note that the CX

4

HX

4

M motif that supplies a Met thioether sulfur ligand, while common (e.g., rusticyanin and hCp), is not found in all T1 Cu sites as illustrated by L494 in Fet3

Figure 2

T1-TNC structural and electronic connectivity

. (a) The overall structural domain of the four Cu sites in an MCO. T1 Cu is shown in blue, T3 Cu in yellow, and T2 Cu in green. The only solvent-exposed Cu site is on the T2 Cu as are the two histidine imidazoles coordinating the T1 Cu; intermolecular electron transfer

into

this Cu prosthetic grouping is

via

these side chains. (b) The electron matrix coupling elements that support intramolecular ET in an MCO are mapped onto the structure by the dashed arrows. These include the backbone of the HCH motif as well as side chain-backbone H-bonding. Although the structures illustrated are from PDB 1zpu, yeast Fet3, one could overlay the same view of any MCO and not tell the difference

Figure 3

MCO cupredoxin domains

. (a) The 3-cupredoxin domain Fet3 protein from

Saccharomyces cerevisiae

(PDB 1zpu). In all structures, the T1 Cu is in blue, the T2 Cu is in green, and the T3 Cu are in yellow. In this view, D1 is to the lower left, D3 is at top. (b) The 6-domain human ceruloplasmin (Cp; PDB 1KCW). The orientation is the same with D1 to the lower left and D6 at the upper left. Note that Cp has T1 sites in D2 and D4, also. (c) The trimer of the 2-domain laccase from

Streptomyces coelicolor

(PDB 1CG8). Note that the formation of the TNC between each dimer and the acquisition of this quaternary structure has a distinct thermodynamic driving force in comparison to the assembly of a TNC in a 3- or 6-domain MCO

Figure 4

Chain connectivity and Cu-site residues in hCp and yeast Fet3

. (a) The four Cu atoms and their coordinating amino acid side chains are shown relative to their positions in the 1046 amino acid polypeptide of the mature human ceruloplasmin. D1 residues are shown in red; D6 residues are shown in black. Note that no sequence-contiguous side chains contribute to the coordination of the same Cu atom with the exception of the cupredoxin, Type 1 Cu motif, CX

4

HX

4

M. The reader should note, however, the multiple examples of HXH motifs where the two His side chains occupy the coordination sphere of different Cu atoms. (b) The canonical distribution of the sequence motifs in either a 3- or 6-domain MCO that contribute to the coordination spheres of the three types of Cu(II). Note that only the C-terminal domain contributes to coordination of the Type 1 Cu and thus represents the ancestral cupredoxin domain that characterizes the small “blue” Cu(II) proteins

Figure 5

Pseudomonas aeruginosa

azurin: holo versus apo structure

. (a,c) Apo-azurin (PDB 1e65) crystallizes as a tetramer; the image in (c) highlights the conformation of the four Type 1 Cu ligands in chain (a) in the absence of the metal ion. (b,d) Holo-azurin (PDB 4azu) adopts a highly comparable overall fold and the four side chains at the Type 1 Cu are essentially unchanged in conformation despite their coordination to the Cu(II)

Figure 6

Coordination sphere of the TNC in Fet3 from

S. cerevisiae

(PDB 1zpu)

. In this orientation, the HCH connectivity is to the right. Note that the two histidine imidazoles are ligand to one each of the T3 Cu atoms. The data in Table 1 indicate that H485 plays a more essential role in maturation of the TNC than does H483; T3

α

and T3

β

are distinguishable within the context of dioxygen reduction at the TNC, as well

Figure 7

Maturation profiles of TNC ligand mutants in Fet3 from

S. cerevisiae

.

The Fet3 ORF encoding the mutant proteins was episomally expressed in

S. cerevisiae

; this clone was terminated prior to the sequence encoding the C-terminal transmembrane domain that tethers wild-type Fet3 to the exo-cytoplasmic surface of the cell and thus these Fet3 species, following full maturation, were secreted into the growth medium, as indicated in the western blot to the right. As Fet3 matures in post-ER compartments, the mannose glycan is elaborated resulting in a steady increase in the apparent molecular mass. In eukaryotes, this is a simple marker of protein trafficking–processing in the secretory pathway

Figure 8

CotA from

Bacillus subtilis

(PDB 1w6l)

. This image highlights the relationship of the T2 Cu relative to the T3 binuclear pair within the groove at the interface between cupredoxin domain 1 (to the right) and domain 3 (to the left). The histidine ligands from these two domains to the T2 Cu in CotA are H105 and H422, respectively. Anaerobic titration of

apo

-CotA with 2 equivalents of Cu

1+

results in complete metallation of the T2 Cu site as well as the T1 one. This could have the effect of constraining D1 relative to D3 in the conformation space needed to populate the binuclear cluster. However, in the TNC, the T2 Cu is “above” the T3 Cu pair and solvent-exposed raising a question about the steric accessibility of the nascent T3 site in a “closed” conformation of this groove

Figure 9

Structure and cellular activity of

Synechococcus

MCO A2319

. (a) In this and other bacterial MCOs, the extended strand that links D2 (top) to D3 (below) in all 3-domain MCOs is clearly seen, appearing like a “basket handle”. In contrast, this strand in Fet3 (PDB 1Zpu) is intercalated into the surface of the two domains. This structure is the work of Drs. Alex Taylor and John Hart (University of Texas Health Science Center, San Antonio) in collaboration with the author. (b) An activity stain of A2319 in the whole cell (WC) and in the isolated cytoplasmic and periplasmic compartments. Solution assay of these latter two fractions indicated that the MCO in each was ∼50% active based on comparison to fully metallated A2319 prepared by subsequent titration with Cu(I). Both assays are based on the “laccase” substrate,

p

-phenylenediamine. In fact, A2319 is a cuprous oxidase, a specificity characteristic of a large cohort of bacterial MCOs (Dr Yun Hee Park and Dr D. J. Kosman, unpublished work)

Assembly of the Redox-Active Metal Centers of Cytochrome

c

Oxidase

Figure 1

Bacterial and mitochondrial cytochrome

c

oxidase (C

c

O): structure and redox metal cofactors

. Panels (a) and (b) show ribbon diagrams of high resolution structures of the four-subunit-

aa

3

-type C

c

O of the

α

-proteobacterium

Rhodobacter sphaeroides

8

(PDB 1M56) and the 13 subunit enzyme from bovine (

Bos taurus

) heart mitochondria

12, 13

(PDB 1OCC). The three-subunit catalytic core of each enzyme is colored the same in each structure. The single-helix subunit IV of the bacterial C

c

O is in gray, as are all of the nucleus-encoded subunits of mitochondrial C

c

O. In panel (c) most of the

R. sphaeroides

protein has been removed to show only the redox-active metal centers, which are essentially identical in the two C

c

Os. A covalent bond between Tyr-288 and the Cu

B

ligand His-284 is present but not shown in this structure. Detailed explanations of the Cu

A

and Cu

B

centers are in the text. These crystal structures of the

R. sphaeroides

and bovine enzymes confirmed that the structure of the catalytic core is similar between prokaryotic and mitochondrial COX

8, 12–16

Figure 2

Heme A biosynthesis and a model for involvement of Coa2, Cox10, Cox15, and Shy1 in mitochondrial Cox1 maturation

. Heme A biosynthesis and COX biogenesis are co-regulated at multiple levels. Heme B is used for heme A biosynthesis in a two-step reaction catalyzed by the heme O synthase Cox10 and subsequently the heme A synthase Cox15, the latter acting in an electron chain with ferredoxin Yah1 and its reductase Arh1. Cox15 acts as a positive regulator of Cox10 function. Heme B levels are sensed by Mss51, a heme-binding protein that acts as a translational activator of COX1 mRNA and a Cox1 chaperone, thereby participating in a negative feedback translation regulation loop. Newly synthesized Cox1 is initially trapped in the Mss51/Cox14/Coa3/Ssc1 intermediate. Coa1, likely in concert with Coa2, triggers further progression of Cox1 to the state where it is competent to receive heme A moieties. Such progression is coupled to Cox10 and Cox15 oligomerization and activation. Incorporation of heme A into the

a

and

a

3

sites culminates in a Shy1-containing intermediate that also contains the Cox5a and Cox6 subunits

Figure 3

Mitochondrial copper metabolism and insertion into Cox1 and Cox2.

The source of copper for C

c

O metallation is a pool of copper in the matrix bound by an ionic ligand (CuL), which in yeast is imported through the Pic2 carrier protein with the help, under stress conditions, of the Mrs3 carrier. Whether these carrier proteins are also involved in copper export to the matrix remains unknown. COX copper metallation occurs in the mitochondrial intermembrane space by C

c

O-specific metallochaperones, COX11 and SCO1 and 2, that each receive Cu(I) from mitochondrial COX17, a Cu(I) binding protein containing a twin CX

9

C motif. At least in yeast, the redox state of a key cysteine and thereby the activity of Cox11 is modulated by the twin CX

9

C protein Cox19 (Section 3.4). In mammals, COX1 is competent for copper metallation in a module that contains the chaperones COX14 and COA3 and the twin CX

9

C protein CMC1. In yeast, copper metallation occurs in a module that additionally contains Shy1 (homolog of SURF1), not depicted here. Insertion of copper into COX2 involves a module containing SCO1 and 2, the COX2 chaperone COX20, and the CX

9

C–CX

10

C protein COA6. Whether or not COA6 plays a direct role in copper delivery remains unclear. The role/s in the process of additional twin CX

9

C proteins, such COX23, remains to be clarified

Figure 4

Model for copper delivery to COX1 by COX11

. The Cox11–Cox11 homodimer (model 5, cluster 2 of Ref. 77) is shown hovering above fully folded Cox1, taken from the structure of the

R. sphaeroides

C

c

O (PDB 1M56). Tethering of the antiparallel dimer by the transmembrane helices of the Cox11 monomers (upper ends are shown in pink) dictates that the four S–two Cu(I) cluster of the Cox11 dimer (Cu(I) ions in red, Cys ligands in blue) will face the membrane surface. Linkers of 15 amino acids between the transmembrane helices and the headgroup of the dimer will keep the Cox11 Cu cluster near the membrane surface. Cu

B

of fully assembled Cox1 is shown in magenta, while the position of the Cu

B

ligands His-333 and His-334 are shown in yellow. Cys-35 of Cox11 is not shown, but Cys-35 of each monomer will be present near the exit of each Cox11 transmembrane helix from the upper surface of the membrane. It can be seen that His-333 and His-334 need to move only a short distance toward the upper surface of Cox1 in order for Cox11 to transfer Cu(I) to these histidines, by the mechanism proposed in Section 3.4

Figure 5

Scenarios of Cu

A

assembly

. The schemes depict five published proposals for the assembly of Cu

A

(explanations are given in Section 4.6)

CuA and CuZ Center Assembly in Nitrous Oxide Reductase

Figure 1 (a) Structure of

Pseudomonas stutzeri

N

2

OR functional homodimer. The backbone of one monomer is represented with the identified secondary structure colored in dark magenta with its transparent surface in pink, and the other monomer is represented as a surface colored in gray. CuA and “CuZ” centers are represented by spheres, in which the copper atoms are colored according to the monomer chain. The distances between CuA and CuZ centers of the two monomers are represented. Figure prepared with Chimera using PDB ID 3SBP. (b) Genome organization of “typical” and “atypical” nosZ gene clusters. Genes are not represented to scale. The identified transcriptional units and promoter regions are identified as arrows and dots, respectively. PA, pseudoazurin; Az, azurin; C, thioredoxin-like protein;

c

,

c

-type cytochrome;

b

,

b

-type cytochrome; FeS, Rieske-like iron–sulfur protein; Fe–S, [4Fe–4S] iron–sulfur protein with a FAD/NAD binding domain. The arrows in black correspond to hypothetical proteins,

dnr

, dissimilative nitrate respiration regulator;

tat

, twin arginine translocation

Figure 2 Structure of CuA and “CuZ” centers of N

2

OR. (a) Structure of CuA and CuZ centers from

Paracoccus denitrificans

N

2

OR, with CuZ center as CuZ*(4Cu1S) in the [1Cu

2+

:3Cu

1+

] oxidation state. The

μ

4

-bridging sulfur is named S

1

, and there is a water/hydroxyl molecule on the Cu

I

–Cu

IV

edge. (b) Structure of CuA and CuZ centers from

Ps. stutzeri

N

2

OR, with CuZ center as CuZ(4Cu2S) in the [2Cu

2+

:2Cu

1+

] oxidation state. The

μ

4

-bridging sulfur is named S

1

, as in (a), and there is an additional sulfur atom, named S

2

, on the Cu

I

–Cu

IV

edge. In this case, CuA

1

atom of CuA center is not coordinated by His583. Figure were prepared in Pymol using PDB ID 1FWX in (a) and PDB ID 3SBP in (b)

Figure 4 Representation of the nitrous oxide reductase maturation process in

Paracoccus denitrificans

, according to the topology of each protein

Figure 3 Topology of the NosZ accessory proteins. The primary sequence of each of the proteins from

Paracoccus denitrificans

was analyzed to determine the transmembrane regions, conserved motif, and other globular domains. TM, transmembrane region (blue rectangles); CASH, carbohydrate-binding proteins and sugar hydrolyses

MoCu CO Dehydrogenase and its Active-Site Assembly

Figure 1 Genetic organization of the

cox

gene cluster on the megaplasmid pHCG3 of

Oligotropha carboxidovorans

OM5. CO dehydrogenase subunits are encoded by

coxMSL

, whereas the protein products of the downstream subcluster

coxDEFG

are functional in the post-translational maturation of the active-site cluster and in anchoring holo-CO dehydrogenase to the cytoplasmic membrane. The whole cluster is transcribed only in the presence of CO

Figure 2 Ribbon representation of the CO dehydrogenase dimer. The subunits are colored differently. Each dimer consists of three different subunits, CoxL, CoxM, and CoxS. Within the structure, the intramolecular electron transport chains are indicated (refer to Section 3.3). Image of 1N5W

6

created with Cn3D (version 4.3.1)

Figure 3 Intramolecular electron transport chain constituted by the CO dehydrogenase subunits CoxL, CoxM, and CoxS. Electrons released in the course of CO oxidation are transferred from the molybdopterin cytosine dinucleotide (MCD) cofactor in CoxL to the [2Fe–2S] clusters in CoxS and subsequently to the FAD in CoxM. Arrows indicate the shortest distances between the cofactors. Image of 1N5W

6

created with Cn3D (version 4.3.1)

Figure 4 CO dehydrogenase active-site cluster. The CoxL subunit harbors a bimetallic [CuSMoO

2

] cluster that catalyzes the oxidation of CO. Molybdenum is coordinated by the ene-dithiolate of the MCD, Cu

1+

is linked to the

γ

-sulfur of Cys

388

. Both metal ions are bridged by a

μ

2

sulfur. MCD stands for molybdopterin cytosine dinucleotide

Figure 5 Structural models for the binding of

n

-butyl-isocyanide (nBIC) (a) and carbon monoxide (b) to the [CuSMoO

2

] active-site cluster of CO dehydrogenase. nBIC is a substrate analogue that binds to the active site in its oxidized state. A thiocarbamate is formed thereby irreversibly inhibiting catalytic activity. Under the assumption that CO binds to the active site in an analogous manner, a thiocarbonate was postulated as potential intermediate in the course of CO oxidation. MCD stands for molybdopterin cytosine dinucleotide

Figure 6 Suggested reactions cycle(s) for the oxidation of CO and potential reaction intermediates. The corresponding structures were postulated with regard to the energy barriers between the states. For more details refer to the text (Section 3.5)

Figure 7 Reaction scheme for the chemical reconstitution of a functional [CuSMoO

2

] active-site cluster. The cycle starts with a nonfunctional [MoO

3

] species (biosynthetic precursor or generated by CN

−

treatment of a functional enzyme) that is first sulfurated. Sulfuration of the molybdenum can occur at both equatorial positions at a 1:1 ratio, but only one position can be reconstituted to a fully assembled [CuSMoO

2

] cluster. After reoxidation and subsequent treatment with a copper-thiourea complex a catalytically active enzyme species is generated

Figure 8 Consensus motifs of the DEAD-box RNA helicase family and the corresponding motif homologues on CoxD, CoxE, and CoxF. DEAD-box RNA helicases consist of a core of at least nine conserved sequence motifs, involved in ATP binding, RNA binding, and ATP hydrolysis. Apart from the Q motif and motif VI, CoxD/E/F reveal homologous sequences for all other motifs

Figure 9 Inactivation of CO dehydrogenase by a thiol compound (R-SH). The latter binds to the [CuSMoO

2

] cluster via its sulfhydryl group generating a 3-coordinate Cu

1+

ion.

37

MCD stands for molybdopterin cytosine dinucleotide

Figure 10 Mo EPR of CO dehydrogenase preparations (12 mg mL

−1

each) in the presence of the sulfur compounds

l

-cysteine (15 mM), sodium sulfide (15 μM), or 2-mercaptoethanol (15 mM). (a) Enzyme from the wild type of

O

.

carboxidovorans

was treated with potassium cyanide and subjected to gel filtration to remove small molecules. Finally, the indicated sulfur compounds were added. (b) Wild type preparations were treated with potassium cyanide and sulfurated by the addition of sodium sulfide and sodium dithionite. After gel filtration, sulfur compounds were added as indicated. (c) The enzyme from the

coxF

mutant (in its as isolated state) was supplied with the indicated sulfur compounds. Spectra were recorded at 120 K at a microwave frequency, modulation amplitude, and microwave power of 9.47 GHz, 1 mT, and 10 mW, respectively. For more details refer to the text (Section 4.3)

Figure 11 Proposed model for the maturation of the bimetallic [CuSMoO

2

] active-site cluster of MoCu CO dehydrogenase. Assembly starts at the [Mo

VI

(=O)

2

OH

(2)

] cluster in the active site of the fully folded apoenzyme. The membrane-bound AAA+ ATPase chaperone CoxD is involved in the sulfuration step of the trioxo molybdenum, leading to a [Mo

VI

(=O)OH

(2)

SH] center. Copper is provided by the cytoplasmic CoxF protein. The latter releases phytate-bound Cu

2+

through its phytase/phosphatase activity and subsequently transfers the metal to its putative copper-binding site. Von Willebrand factor A (vWA) interactions enable complex formation between membrane-bound CoxE and soluble CoxF, thereby attaching CoxF to the cytoplasmic membrane. Electrons from the respiratory electron transport system (ETS) are used to reduce Cu

2+

to Cu

1+

, which is afterward inserted into the [Mo

VI

(=O)OH

(2)

SH] cluster, yielding a fully assembled [Mo(=O)OH-S-Cu-S-Cys] active site

Figure 12 Recruitment of soluble CO dehydrogenase to the inner surface of the cytoplasmic membrane. Characterization of CO dehydrogenase preparations from the

coxG

mutant indicates that maturation of the [CuSMoO

2

] active-site cluster is complete at the stage of

coxG

. Sequence analyses for CoxG revealed the presence of a pleckstrin homology (PH) domain, which suggests that CoxG could be functional in recruiting the fully assembled, soluble CO dehydrogenase to the inner aspect of the cytoplasmic membrane thereby enabling electron transfer to the quinone pool and the respiratory chain. The ability to use CO as sole carbon and energy source was impaired in the

coxG

mutant, which could be ascribed to an insufficient interaction of CO dehydrogenase with the membrane

Homo- and Heterometallic Dinuclear Manganese Proteins: Active Site Assembly

Figure 1

The ferritin-like superfamily.

(a) Schematic view of the relationships between the major ferritin-like protein families and their dinuclear metal clusters. Common branch color indicates shared monomer topology:

tan

, the simplest topology consisting only of the four-helix bundle;

yellow

, the Mn-catalase topology;

green

, the core of the topology shared by substrate-oxidizing enzymes and RNR radical-generating subunits. (b) Schematic four-helix bundle fold with ligand positions marked (red: carboxylates, dark red: histidine). The inset is based on the

Desulfovibrio desulfuricans

bacterioferritin structure (PDB 1nfv; PDB—Protein Data Bank)

Figure 2

Overview of selected manganese-containing dinuclear metallocofactors.

(a)

Canavalia ensiformis

concanavalin A (PDB 1jbc), (b) human arginase (PDB 2aeb), (c) human prolidase (PDB 2okn). Coordinating amino acids displayed as sticks. Color coding: C: gray; N: blue; O: red; B: pink; P: yellow; Ca: green; Mn: purple

Figure 3

Intracellular ratio between manganese and iron in different organisms.

Figure 4

Schematic RNR reaction mechanism

. Ribonucleotide reduction takes place in four basic steps, the first of which involves activation of the substrate through abstraction of a hydrogen atom at the 3′ position of the ribose by a cysteinyl radical (Cys

•

). Subsequently, the 2′ OH group leaves as water, the substrate is reduced with two electrons, and the initially abstracted 3′ hydrogen is returned to the substrate to form the complete product. In class I RNRs the metallocofactor in the β subunit generates the initial transient Cys

•

via a reversible long-range radical transfer

Figure 5

Maximum likelihood phylogeny of class I RNR β subunits.

Sequences representing the total diversity of class I RNR β subunits were selected and aligned and the phylogeny estimated with FastTree.

23

The unrooted tree is shown with NrdF as outgroup. The traditional subclassification based on biochemical characteristics is indicated for subclasses Ib–Id with dashed boxes. A phylogenetic subclassification of β subunits performed by us is indicated by colored clades and explained by colored labels in the leftmost column. In the middle column assumed metal centers are indicated. Evidence for the composition of metal centers is only present for a few enzymes (e.g.,

E. coli

NrdBg and NrdF,

Bacillus

spp.,

Corynebacterium ammoniagenes

and

Streptoccocus

spp. NrdF,

Dictyostelium discoideum

,

Saccharomyces cerevisiae

,

Mus musculus

and

Homo sapiens

NrdBe,

Pseudomonas aeruginosa

NrdBz (Fe

2

),

Chlamydia trachomatis

and

Saccharopolyspora erythraea

NrdBz (MnFe; subclass Ic), and

Flavobacterium johnsoniae

NrdBi). The composition of the majority of other enzyme's metal centers was estimated based on the phylogeny and sequence comparison. The NrdF subclass contains several sequences with non-canonical ligands, suggesting metal centers other than the Mn

2

indicated in the Figure may be present. The distribution among organisms of each subclass is indicated by differently sized circles in the rightmost column. No attempt at correction of the influence of bias in the sequencing of bacterial genomes was performed

Figure 6

Overview of the mechanism and active site structures of Mn catalase.

(a) Schematic reaction mechanism of Mn catalase; (b–d) the active sites of manganese-containing catalases from

L. plantarum

(PDB 1jku) (b),

T. thermophilus

(PDB 2v8u) (c), and KatB from

Anabaena

(PDB 4r42) (d). Coordinating amino acids displayed as sticks. Color coding: C: gray; N: blue; O: red; Mn: purple

Figure 7

Ferritin-like superfamily sequence logos

. Coordinating amino acids indicated by black arrows and the position of the radical harboring tyrosine residue in RNR β subunits by a green arrow. Complete conservation is indicated by single letters and information content is indicated by height of letters. Low degree of conservation, for example, for the fifth ligand in Mn catalase and the first ligand and the position of the tyrosine radical in NrdBz, is shown by combinations of letters with lower height

Figure 8

The active site of

C. ammoniagenes

NrdF

. Coordinating amino acids and the radical harboring tyrosine residue (Y115) displayed as sticks. Color coding: C: gray; N: blue; O: red; Mn: purple. (PDB 3mjo)

Figure 9

Overview of the assembly of the Mn

III

2

-Tyr

•

cofactor in NrdF

. (a) Schematic summary of the activation mechanism. A superoxide radical, generated by NrdI, oxidizes the Mn

II

2

precursor to generate the catalytically relevant Mn

III

2

-Tyr

•

cofactor via an Mn

III

Mn

IV

intermediate. (b) The NrdI–NrdF channel in the NrdI–NrdF complex extending from the NrdF active site to the FMN cofactor. The complex channel is depicted as a light blue mesh and was calculated using a 1.4 Å probe radius. Selected NrdI (green) and NrdF (white) residues lining the channel are shown as sticks. (From A. K. Boal, J. A. Cotruvo, J. Stubbe, and A. C. Rosenzweig, Science, 2010, 329, 1526–30. Reprinted with permission from AAAS). (c) Reaction pathway for NrdI-catalyzed formation of the NrdF activating superoxide radical from NrdI

sq

. In some species NrdI

hq

appears to be the catalytically relevant state, which can generate either two equivalents of the active O

2

•−

species (right) or regenerate NrdI

q

through formation of one equivalent of the unproductive H

2

O

2

(left, dashed line). Note that the scheme is not balanced with regard to protons.

Figure 10

Overview of the active sites of Mn

II

2

-NrdF proteins.

(a)

E. coli

(PDB 3mjo); (b)

B. subtilis

(PDB 4dr0); and (c)

B. cereus

(PDB 4bmu). Coordinating amino acids and the radical harboring tyrosine residue displayed as sticks. Color coding: C: gray; N: blue; O: red; Mn: purple

Figure 11

The active site of sweet potato PAP

. Coordinating amino acids displayed as sticks. Color coding: C: gray; N: blue; O: red; P: yellow; Mn: purple; Fe: orange. (PDB 1xzw)

Figure 12

The active site of

C. trachomatis

R2c in different oxidation states.

(a) In reduced state (PDB 4m1i), and (b) oxidized state (PDB 1syy). It should be noted that the structure of the oxidized state of the active site is obtained with diiron. Coordinating amino acids and the phenylalanine, replacing the radical harboring tyrosine, residue displayed as sticks. Color coding: C: gray; N: blue; O: red; Mn: purple; Fe: orange

Figure 13

Apo- and Mn

II

Fe

II

-R2c.

Highly similar positioning of the ligating amino acids suggest a very rigid coordination environment. Color coding: C: gray; N: blue; O: red; Mn: purple; Fe: orange. Metal sites 1 and 2 indicated with numbers in bold (

1

and

2

).

Figure 14

Assembly of the metallocofactor of R2c.

In the presence of stoichiometric amounts of Mn

II

and Fe

II

, the Mn

II

Fe

II

precursor assembles and is spontaneously oxidized to the catalytically relevant Mn

IV

Fe

III

cofactor under aerobic conditions proceeding via a Mn

IV

Fe

IV

intermediate. The presence of excess Fe

2+

results in the formation of a Fe

IV

Fe

III

complex in the presence of oxygen, while the Mn

II

2

complex remains in its low-valent state

Figure 15

The active site of R2lox in different oxidation states

. (a) In reduced (Mn

II

Fe

II

) state (PDB 4hr4) and (b) oxidized (Mn

III

Fe

III

) state (PDB 4hr0). Coordinating amino acids and the metal-bound fatty acid displayed as sticks. Color coding: C: gray; N: blue; O: red; Mn: purple; Fe: orange

Figure 16

Schematic overview of the activation of R2lox.

The binding of Mn

II

in site 1 is preceded by Fe

II

binding in site 2. Oxidation by O

2

presumably generates an Mn

IV

Fe

IV

intermediate, which in turn oxidizes two nearby amino acids, resulting in a valine–tyrosine cross-link, with concomitant reduction of the metal center to its stable Mn

III

Fe

III

state

Biogenesis and Assembly of the CaMn

4

O

5

Core of Photosynthetic Water Oxidases and Inorganic Mutants

Figure 1 Two views of monomeric PSII. (a) PSII as viewed through the thylakoid membrane, with the stromal side at the top and luminal side at the bottom. (b) PSII as viewed from the lumen. Several major subunits have been marked, as well as the location of the water-oxidizing complex (WOC) and the original location of small subunits displaced by crystallization technique

7

.

Figure 2 (a) The WOC and its coordinating residues, as obtained from the crystal structure by Umena

et al

.

10

Directions of channels allowing access to the lumen are marked. (Reproduced with permission from Ref.

10

. © Nature Publishing Group, 2011.) (b) The structure around tyrosine-Z (Y

z

, D1-Y161).

Figure 3 (a) PSII monomer showing 103 arginines (red) and internal channels (red, blue, and yellow tubes) after Guskov

et al

.

18

Numerous arginines are found around the channels, and one, CP43-R357, even coordinates Mn#4 in the WOC itself. (Reproduced with permission from Ref.

18

. © Nature Publishing Group, 2009.) (b) Chemical structure of arginine-bicarbonate ion pairing and function in H

+

neutralization and CO

2

buffering for terminal reaction (Calvin cycle)

Figure 4 Stability fields of manganese species in aqueous solution in the pH range from 4.0 to 10.0. The solution contains no sulfate. Dissolved manganese activity ranges from 0.01 to 100 ppm at a constant partial pressure of CO

2

= 3.8 × 10

−4

atmosphere

Figure 5 Stoichiometry of inorganic cofactors during photoassembly and their relationship to the atomic structure determined by XRD

10

. (Reproduced with permission from Ref.

10

. © Nature Publishing Group, 2011.)

Figure 6 Photoassembly mechanism and intermediates through IM

1

* as determined to date (a) and spectroscopically deduced chemical structures from EPR/ENDOR/ESEEM (b)

Figure 7 Charge transfer equilibrium between the WOC(Ca) and Y

z

+

changes upon replacing Ca by Sr, favoring hole transfer into the WOC(Sr) and resulting in greater O

2

quantum yield

34

. (Reproduced from Ref.

34

© Elsevier 2012.)

Figure 8 Determination of the number of single turnover laser flashes needed to produce the first detectable O

2