Metalloprotein Active Site Assembly

Michael K. Johnson, Robert A. Scott, Michael K. Johnson, Robert A. Scott

• Publisher: Wiley
• Year: 2017
• Print ISBN: 9781119159834
• eBook ISBN: 9781119159858
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Inorganic Chemistry

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.

Part 1

Assembly and Trafficking of Simple Fe-S Clusters

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

Patricia C. Dos Santos

Wake Forest University, Winston-Salem, NC, USA

and

Dennis R. Dean

Virginia Tech, Blacksburg, VA, USA

1. 1 Introduction
2. 2 The Nif System as a Model for Analysis of Simple [Fe–S] Cluster Assembly
3. 3 Analysis of the Mo-Dependent Nitrogen-Fixing System in A. vinelandii
4. 4 Genetic Phenotypes and Biochemical Features Indicated a Role for NifU and NifS in [Fe–S] Cluster Formation
5. 5 NifS Cysteine Desulfurase
6. 6 NifU Provides a Scaffold for [Fe–S] Cluster Assembly
7. 7 NifU and NifS as the Minimum Set for the Assembly and Transfer of Fe–S Clusters
8. 8 The NifS/NifU [Fe–S] Cluster Assembly Toolkit Provides a Paradigm for Simple [Fe–S] Cluster Assembly
9. 9 Functional Cross Talk Between [Fe–S] Cluster Biosynthetic Systems
10. 10 Concluding Remarks
11. 11 Acknowledgments
12. 12 Related Articles
13. 13 Abbreviations and Acronyms
14. 14 References

1 Introduction

In this chapter, biological iron–sulfur [Fe–S] clusters are defined as any protein-bound prosthetic group that contains Fe and S, and releases H₂S when treated with acid.^{1, 2} Owing to their structural and chemical versatilities [Fe–S] clusters participate in a very large number of key biological processes, including electron transfer, substrate activation, DNA synthesis and repair, sulfur mobilization, Fe storage, and gene regulation.^2–4 Indeed, [Fe–S] clusters are essential players in a wide range of life-sustaining processes. The most prevalent [Fe–S] clusters found in nature are [2Fe–2S] and [4Fe–4S] (Figure 1), which are attached most frequently to their cognate protein partners through cysteine residue thiol ligands. However, other coordination types, either N-ligands provided by a histidine residue or carboxylate ligands provided by an aspartate residue, have also been described.³ In addition to simple [Fe–S] clusters, there are also more complex [Fe–S] clusters having a higher nuclearity, for example the [8Fe–7S] P cluster contained within nitrogenase.⁵ Even more complex [Fe–S] clusters are those that contain a different metal in addition to Fe and also an organic constituent, for example the suite of active site cofactors contained in the nitrogenases described later (Figure 1).

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)

A basic architecture constructed from simple [2Fe–2S] and/or [4Fe–4S] cluster units is a common feature of most complex [Fe–S] clusters.² Simple [2Fe–2S] and [4Fe–4S] clusters can be readily formed in the laboratory under anoxic and reducing conditions by mixing Fe and S in appropriate concentrations in the presence of suitable ligands.⁶ However, the biological process is complicated by the fact that free Fe²⁺ and free S²⁻ are highly toxic to living cells. Thus, a basic question that has challenged the bioinorganic research community is: how are simple [2Fe–2S] and [4Fe–4S] clusters formed biologically?

A common approach toward probing fundamental biological processes is to characterize mutants impaired in some aspect of the process in order to identify key components. Once participating components are identified by genetic means, biochemical and biophysical analyses can be applied in efforts to duplicate the process using purified components. This approach is often an iterative one wherein genetic manipulations inform biochemical experiments, the outcome of which, in turn, suggests further genetic manipulation, and vice versa. However, for such a process to be useful several criteria must be met. First, the experimental organism must be amenable to genetic manipulation. Second, it must be possible to correlate genetic defects with perturbations in the process under study. Third, the identified components must be obtained in sufficient levels to permit structural and functional interrogation.

It might seem remarkable that an understanding of the biosynthetic pathways for the formation of many highly complex organic cofactors far preceded the discovery of mechanistic features associated with biological [Fe–S] cluster formation. Timely identification of key aspects of [Fe–S] cluster assembly that paralleled elucidation of the pathways for formation of many organic cofactors was denied for several important reasons. Namely, owing to the structural simplicity of [2Fe–2S] and [4Fe–4S] clusters, as well as the development of facile methods for their robust chemical synthesis, the process did not capture the interest of the scientific community until relatively recently. Also, because [Fe–S] clusters are essential to so many biological processes, the fortuitous discovery of a particular metabolic defect that could be unambiguously assigned to a perturbation in [Fe–S] cluster assembly did not occur. In this chapter, we describe why components involved in producing an active nitrogenase, the catalytic component of biological nitrogen fixation, proved to be a useful experimental system for the study of simple [Fe–S] cluster assembly. We also describe how this experimental system was used to discover some of the key principles of biological [Fe–S] cluster assembly.

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

Nitrogenase is an enzyme found only in a specialized class of microorganisms called diazotrophs (nitrogen eaters). It catalyzes biological nitrogen fixation, the reduction of N₂ to ammonia. The most thoroughly studied diazotroph is Azotobacter vinelandii. This organism has three distinct but structurally and mechanistically similar systems that are capable of nitrogen fixation.^7–10 A striking similarity among the three systems is that the catalytic entity for each is comprised of two component proteins, neither of which has the capacity to reduce N₂ in the absence of the other. One component serves as a nucleotide-depende...

Table of contents

• Cover
• Title Page
• Copyright
• Table of Contents
• 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
• Part 2: Assembly of Complex and Heterometallic Fe-S Cluster Active Sites
• Part 3: Assembly of Homometallic and Heterometalic Cu Cluster Active Sites
• Part 4: Assembly of Homometallic and Heterometallic Mn Clusters
• Part 5: Assembly of Homometallic and Heterometallic Ni Clusters
• Part 6: Assembly of Cofactors for Binding Active-site Metal Centers
• Index
• Abbreviations and Acronyms used in this Volume
• End User License Agreement