Saumen Chakraborty

Metalloproteins containing cytochrome, iron-sulfur, or copper redox centers.

Redox reactions play important roles in almost all biological processes, including photosynthesis and respiration, which are two essential energy processes that sustain all life on earth. It is thus not surprising that biology employs redox-active metal ions in these processes. It is largely the redox activity that makes metal ions uniquely qualified as biological cofactors and makes bioinorganic enzymology both fun to explore and challenging to study. Even though most metal ions are redox active, biology employs a surprisingly limited number of them for electron transfer (ET) processes. Prominent members of redox centers involved in ET processes include cytochromes, iron–sulfur clusters, and cupredoxins. Together these centers cover the whole range of reduction potentials in biology (Figure ​(Figure1).1). Because of their importance, general reviews about redox centers1−77 and specific reviews about cytochromes,8,24,78−90 iron–sulfur proteins,91−93 and cupredoxins94−104 have appeared in the literature. In this review, we provide both classification and description of each member of the above redox centers, including both native and designed proteins, as well as those proteins that contain a combination of these redox centers. Through this review, we examine structural features responsible for their redox properties, including knowledge gained from recent progress in fine-tuning the redox centers. Computational studies such as DFT calculations become more and more important in understanding the structure–function relationship and facilitating the fine-tuning of the ET properties and reduction potentials of metallocofactors in proteins. Since this aspect has been reviewed extensively before,105−110 and by other reviews in this thematic issue,2000,2001,2002 it will not be covered here. Figure 1 Reduction potential range of redox centers in electron transfer processes.

Design of a Three‐Helix Bundle Capable of Binding Heavy Metals in a Triscysteine Environment

An important objective of de novo protein design is the preparation of metalloproteins, as many natural systems contain metals that play crucial roles for the function and/or structural integrity of the biopolymer. Metalloproteins catalyze some of the most important processes in nature, from energy generation and transduction to complex chemical transformations. At the same time, metals in excess can be deleterious to cells, and some ions are purely toxic, with no known beneficial effects (e.g., Hg or Pb). Ideally, we would hope to be able to use an approach based on first principles to create both known metallocenters and novel sites, which may lead to exciting new catalytic transformations. However, the design of novel metalloproteins is a challenging and complex task, especially if the aim is to prepare asymmetric metal environments. Numerous metalloprotein systems have been designed over the past 15 years, typically through the use of unassociated peptides that assemble into three-stranded coiled coils or helix–loop–helix motifs that form antiparallel fourstranded bundles. In terms of metal-ion binding, these systems have been functionalized with heme and nonheme mononuclear and binuclear centers. It is often difficult to prepare nonsymmetrical metal sites through these strategies owing to the symmetry of the systems, which rely on homooligomerization. Thus, the preparation of a single polypeptide chain capable of controlling a metal-coordination environment is a key objective. Previously, we designed soft, thiol-rich metal-binding sites involving cysteine and/or penicillamine as the ligating amino acid residues into the interior of parallel, three-stranded ahelical coiled coils. These systems have served as hallmarks for understanding the metallobiochemistry of different heavy metals, such as Cd, Hg, As, and Pb. We have shown how to control the geometry and coordination number of metals such as Cd and Hg at the protein interior and how to fine-tune the physical properties of the metals, which led to site-selective molecular recognition of Cd. Although these homotrimeric assemblies have been very useful, the production of heterotrimeric systems in which metal environments could be fine-tuned controllably or a hydrogen bond could be introduced site-specifically has been elusive. Therefore, we chose an alternative strategy to satisfy this objective and used a single polypeptide chain instead of multiple self-associating peptides. Existing designed heteromeric helical bundles and coiled coils show energetic preferences of several kcalmol 1 for the desired heteromeric versus homomeric assemblies. However, the energy gap between a heteroand homomeric assembly often depends critically on ionic strength, the pH value, and other environmental parameters. Moreover, the objective of many studies in de novo protein design is to make the metal ion adopt an energetically suboptimal coordination geometry, and the degree to which this strategy will be successful depends on the size of the energy gap between the desired heteromeric assembly and other homomeric or misfolded states. Also, even when heterooligomeric bundles have been used to successfully identify specific environmental effects that influence substrate binding or the reactivity of a metal-ion cofactor, the noncovalently assembled complexes have often been difficult to characterize structurally, possibly owing to small populations of alternatively assembled species. In this case, the inclusion of the active-site residues in a construct with linked helices greatly facilitated structural analysis and catalytic characterization. An attractive starting scaffold to meet our objectives is the de novo designed three-helix bundle a3D. The structure of this protein has been determined by NMR spectroscopy, and it has been proven that the helices are oriented in a counterclockwise topology. Although the a3D protein originated from a coiled coil, its helices were shortened to such an extent that it might be better considered as a globular protein whose repetitive structure makes each of the heptads very similar to one another (in the absence of end effects). The stability of a3D is similar to that of natural proteins. Thus, a3D should be tolerant to mutations, and this protein should serve as an excellent framework for the engineering of specific metalbinding sites. Additionally, with this protein scaffold, we can study the effect of the ligating residue located on the second [*] S. Chakraborty, Dr. J. Yudenfreund Kravitz, Prof. V. L. Pecoraro Department of Chemistry, University of Michigan Ann Arbor, MI 48109 (USA) Fax: (+1)734-936-7628 E-mail: vlpec@umich.edu

A Hybrid DNA-Templated Gold Nanocluster For Enhanced Enzymatic Reduction of Oxygen

We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ∼1 nm in diameter and possessing ∼7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ∼15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e(-) reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (FeBMbs) Proceeds through a trans-Iron-Nitrosyl Dimer

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N2O. Engineering a nonheme FeB site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N2O in solution and to show that the presence of a distal FeBII is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}7 species with ν(FeNO)heme and ν(NO)heme at 522 and 1660 cm–1, and a second NO molecule is bound to the nonheme FeB site with a ν(NO)FeB at 1755 cm–1. Stopped-flow UV–vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to FeB is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex with characteristic ν(FeNO)heme at 570 ± 2 cm–1 and ν(NO)FeB at 1755 cm–1. Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in FeBMb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex to decay with production of N2O at a rate of 0.7 s–1 at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs.

Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases

Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for natures choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

Spectroscopic and Computational Study of a Nonheme Iron Nitrosyl Center in a Biosynthetic Model of Nitric Oxide Reductase

A major barrier to understanding the mechanism of nitric oxide reductases (NORs) is the lack of a selective probe of NO binding to the nonheme FeB center. By replacing the heme in a biosynthetic model of NORs, which structurally and functionally mimics NORs, with isostructural ZnPP, the electronic structure and functional properties of the FeB nitrosyl complex was probed. This approach allowed observation of the first S=3/2 nonheme {FeNO}(7) complex in a protein-based model system of NOR. Detailed spectroscopic and computational studies show that the electronic state of the {FeNO}(7) complex is best described as a high spin ferrous iron (S=2) antiferromagnetically coupled to an NO radical (S=1/2) [Fe(2+)-NO(.)]. The radical nature of the FeB -bound NO would facilitate N-N bond formation by radical coupling with the heme-bound NO. This finding, therefore, supports the proposed trans mechanism of NO reduction by NORs.

Probing the Coordination Environment of the Human Copper Chaperone HAH1: Characterization of HgII-Bridged Homodimeric Species in Solution

Although metal ion homeostasis in cells is often mediated through metallochaperones, there are opportunities for toxic metals to be sequestered through the existing transport apparatus. Proper trafficking of Cu(I) in human cells is partially achieved through complexation by HAH1, the human metallochaperone responsible for copper delivery to the Wilson and Menkes ATPase located in the trans-Golgi apparatus. In addition to binding copper, HAH1 strongly complexes Hg(II), with the X-ray structure of this complex previously described. It is important to clarify the solution behavior of these systems and, therefore, the binding of Hg(II) to HAH1 was probed over the pH range 7.5 to 9.4 using (199)Hg NMR, (199m)Hg PAC and UV-visible spectroscopies. The metal-dependent protein association over this pH range was examined using analytical gel-filtration. It can be concluded that at pH 7.5, Hg(II) is bound to a monomeric HAH1 as a two coordinate, linear complex (HgS2), like the Hg(II)-Atx1 X-ray structure (PDB ID: 1CC8). At pH 9.4, Hg(II) promotes HAH1 association, leading to formation of HgS3 and HgS4 complexes, which are in exchange on the μs-ns time scale. Thus, structures that may represent central intermediates in the process of metal ion transfer, as well as their exchange kinetics have been characterized.

Recent Advances in Biosynthetic Modeling of Nitric Oxide Reductases and Insights Gained from Nuclear Resonance Vibrational and Other Spectroscopic Studies

This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth’s nitrogen cycle where they catalyze the proton-dependent two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV–vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reaction of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent 57Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. The outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.

3.19 – Metalloprotein Design

Metalloproteins catalyze numerous biological reactions ranging from photosynthesis, respiration, nitrogen fixation to signal transduction and complex chemical reactions. It is thus not surprising that metalloproteins account for almost one-half of the total number of proteins in nature. A considerable effort has been directed toward understanding the structure–function relationships using native proteins. However, it is an ultimate challenge to design metalloproteins using only the minimal features required to reproduce their functionalities as well as confer them with novel and unprecedented functionalities learned from the design process. In this chapter, we review some recent successes in the field of metalloprotein design using either de novo designed or native protein scaffolds. Furthermore, metalloprotein design employing unnatural amino acids or non-native cofactor are summarized. Finally, methodologies employing rational design, combinatorial selection, or both methods are also discussed.

Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron–Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (FeBMbs): Insights into the Mechanism of Denitrifying NO Reductases

Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.