Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the Vibrio cholerae MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) Protein Sci. 19, 1830–1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides.

Zinc-substituted cytochrome c (Zn-cyt c) is noncovalently bound to single-walled carbon nanotubes (SWNTs), causing the Zn-cyt c fluorescence to be quenched by up to 95%, primarily due to photoinduced charge transfer. Deposition of Zn-cyt c/SWNT films onto conductive oxides allows for harvesting of photoexcited electrons with an internal quantum efficiency of over 5%.

There has been great interest in the development of stable, inexpensive, efficient catalysts capable of reducing aqueous protons to hydrogen (H2), an alternative to fossil fuels. While synthetic H2 evolution catalysts have been in development for decades, recently there has been great progress in engineering biomolecular catalysts and assemblies of synthetic catalysts and biomolecules. In this Forum Article, progress in engineering proteins to catalyze H2 evolution from water is discussed. The artificial enzymes described include assemblies of synthetic catalysts and photosynthetic proteins, proteins with cofactors replaced with synthetic catalysts, and derivatives of electron-transfer proteins. In addition, a new catalyst consisting of a thermophilic cobalt-substituted cytochrome c is reported. As an electrocatalyst, the cobalt cytochrome shows nearly quantitative Faradaic efficiency and excellent longevity with a turnover number of >270000.

The cobalt complex of an amino-terminal copper and nickel (ATCUN) motif model tripeptide (CoGGH) is shown to act as an electrocatalyst for hydrogen evolution from water near neutral pH with high Faradaic efficiency. The catalyst performance is not significantly impacted by exposure to oxygen. CoGGH represents a new class of hydrogen evolution catalyst that is straightforward to prepare and to modify.

Carbon paste paper electrodes (CPPEs) were fabricated by coating a regular paper strip with carbon paste made from graphite powder and mineral oil, followed by coating with polyaniline. The CPPEs were evaluated as anodes in bioelectrochemical cells (BECs) using Shewanella oneidensis MR-1 as bacteria that donate electrons through extracellular electron transfer. The CPPE was compared to a carbon felt electrode (CFE) modified with polyaniline under the same conditions. The BEC using the CPPE anode produces current continuously for at least 4 days without the need for additional fuel (lactate). Twenty-four hours after inoculation, the BEC using the CPPE anode generates a current density more than two times greater than that of the cell using the CFE, with a competitive maximum value of 2.2 A m–2. The simple fabrication, ease of modification, and low cost of the CPPE make it a promising new bioelectrode material for microbial fuel cells.

Hemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme c, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme c properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented.Proteins that bind heme c are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes c (cyts c) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts c, the functional significance of heme covalent attachment has remained elusive. One observation is that heme c reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme b, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts c and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme c lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt c. Another consequence of heme covalent attachment is the strong vibrational coupling seen between the iron and the protein surface as revealed by nuclear resonance vibrational spectroscopy studies. Finally, heme covalent attachment is proposed to be an important feature supporting multiple roles of cyt c in programmed cell death (apoptosis).Heme covalent attachment is not only vital for the biological functions of cyt c but also provides a useful handle in a number of applications. For one, the engineering of heme c onto an exposed portion of a protein of interest has been shown to provide a visible affinity purification tag. In addition, peptides with covalently attached heme, known as microperoxidases, have been studied as model compounds and oxidation catalysts and, more recently, in applications for energy conversion and storage. The wealth of insight gained about heme c through fundamental studies of cyts c forms a basis for future efforts toward engineering natural and artificial cytochromes for a variety of applications.

Cytochrome c (Cyt c) has a heme covalently bound to the polypeptide via a Cys-X-X-Cys-His (CXXCH) linker that is located in the interface region for protein–protein interactions. To determine whether the polypeptide matrix influences iron vibrational dynamics, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c-552, and variants M13V, M13V/K22M, and A7F, which have structural modifications that alter the composition or environment of the CXXCH pentapeptide loop. Simulations of the NRVS data indicate that the 150–325 cm–1 region is dominated by NHis–Fe–SMet axial ligand and polypeptide motions, while the 325–400 cm–1 region shows dominant contributions from ν(Fe–NPyr) (Pyr = pyrrole) and other heme-based modes. Diagnostic spectral signatures that directly relate to structural features of the heme active site are identified using a quantum chemistry-centered normal coordinate analysis (QCC-NCA). In particular, spectral features that directly correlate with CXXCH loop stiffness, the strength of the Fe–His interaction, and the degree of heme distortion are identified. Cumulative results from our investigation suggest that compared to the wild type (wt), variants M13V and M13V/K22M have a more rigid CXXCH pentapeptide segment, a stronger Fe–NHis interaction, and a more ruffled heme. Conversely, the A7F variant has a more planar heme and a weaker Fe–NHis bond. These results are correlated to the observed changes in reduction potential between wt protein and the variants studied here. Implications of these results for Cyt c biogenesis and electron transfer are also discussed.

A molecular electrocatalyst is reported that reduces protons to hydrogen (H2) in neutral water under aerobic conditions. The biomolecular catalyst is made from cobalt substitution of microperoxidase-11, a water-soluble heme-undecapeptide derived from the protein horse cytochrome c. In aqueous solution at pH 7.0, the catalyst operates with near quantitative Faradaic efficiency, a turnover frequency ∼6.7 s–1 measured over 10 min at an overpotential of 852 mV, and a turnover number of 2.5 × 104. Catalyst activity has low sensitivity to oxygen. The results show promise as a hydrogenase functional mimic derived from a biomolecule.

The heme in cytochromes c undergoes a conserved out-of-plane distortion known as ruffling. For cytochromes c from the bacteria Hydrogenobacter thermophilus and Pseudomonas aeruginosa, NMR and EPR spectra have been shown to be sensitive to the extent of heme ruffling and to provide insights into the effect of ruffling on the electronic structure. Through the use of mutants of each of these cytochromes that differ in the amount of heme ruffling, NMR characterization of the low-spin (S = 1/2) ferric proteins has confirmed and refined the developing understanding of how ruffling influences the spin distribution on heme. The chemical shifts of the core heme carbons were obtained through site-specific labeling of the heme via biosynthetic incorporation of 13C-labeled 5-aminolevulinic acid derivatives. Analysis of the contact shifts of these core heme carbons allowed Fermi contact spin densities to be estimated and changes upon ruffling to be evaluated. The results allow a deconvolution of the contributions to heme hyperfine shifts and a test of the influence of heme ruffling on the electronic structure and hyperfine shifts. The data indicate that as heme ruffling increases, the spin densities on the β-pyrrole carbons decrease while the spin densities on the α-pyrrole carbons and meso carbons increase. Furthermore, increased ruffling is associated with stronger bonding to the heme axial His ligand.

Analysis of NMR spectra reveals that the heme axial Met ligand orientation and dynamics in Nitrosomonas europaea cytochrome c552 (Ne cyt c) are dependent on the heme redox state. In the oxidized state, the heme axial Met is fluxional, interconverting between two conformers related to each other by inversion through the Met δS atom. In the reduced state, there is no evidence of fluxionality, with the Met occupying one conformation similar to that seen in the homologous Pseudomonas aeruginosa cytochrome c551. Comparison of the observed and calculated pseudocontact shifts for oxidized Ne cyt c using the reduced protein structure as a reference structure reveals a redox-dependent change in the structure of the loop bearing the axial Met (loop 3). Analysis of nuclear Overhauser effects (NOEs) and existing structural data provides further support for the redox state dependence of the loop 3 structure. Implications for electron transfer function are discussed.

It is shown that cytochrome c heme lyase (CCHL) attaches heme covalently to peptides composed of the N-terminal segment of cyt c fused to a non-heme containing protein, lending insight into the substrate specificity of CCHL and providing a new route to artificial heme proteins.

The active site of cytochrome c (Cyt c) consists of a heme covalently linked to a pentapeptide segment (Cys-X-X-Cys-His), which provides a link between the heme and the protein surface, where the redox partners of Cyt c bind. To elucidate the vibrational properties of heme c, nuclear resonance vibrational spectroscopy (NRVS) measurements were performed on 57Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c552, including 13C8-heme–, 13C515N-Met–, and 13C15N-polypeptide (pp)–labeled samples, revealing heme-based vibrational modes in the 200- to 450-cm−1 spectral region. Simulations of the NRVS spectra of H. thermophilus cytochrome c552 allowed for a complete assignment of the Fe vibrational spectrum of the protein-bound heme, as well as the quantitative determination of the amount of mixing between local heme vibrations and pp modes from the Cys-X-X-Cys-His motif. These results provide the basis to propose that heme-pp vibrational dynamic couplings play a role in electron transfer (ET) by coupling vibrations of the heme directly to vibrations of the pp at the protein–protein interface. This could allow for the direct transduction of the thermal (vibrational) energy from the protein surface to the heme that is released on protein/protein complex formation, or it could modulate the heme vibrations in the protein/protein complex to minimize reorganization energy. Both mechanisms lower energy barriers for ET. Notably, the conformation of the distal Met side chain is fine-tuned in the protein to localize heme-pp mixed vibrations within the 250- to 400-cm−1 spectral region. These findings point to a particular orientation of the distal Met that maximizes ET.

Electron paramagnetic resonance (EPR) spectra of variants of Hydrogenobacter thermophilus cytochrome c552 (Ht c-552) and Pseudomonas aeruginosa cytochrome c551 (Pa c-551) are analyzed to determine the effect of heme ruffling on ligand-field parameters. Mutations introduced at positions 13 and 22 in Ht c-552 were previously demonstrated to influence hydrogen bonding in the proximal heme pocket and to tune reduction potential (Em) over a range of 80 mV [Michel, L. V.; Ye, T.; Bowman, S. E. J.; Levin, B. D.; Hahn, M. A.; Russell, B. S.; Elliott, S. J.; Bren, K. L. Biochemistry2007, 46, 11753–11760]. These mutations are shown here to also increase heme ruffling as Em decreases. The primary effect on electronic structure of increasing heme ruffling is found to be a decrease in the axial ligand-field term Δ/λ, which is proposed to arise from an increase in the energy of the dxy orbital. Mutations at position 7, previously demonstrated to influence heme ruffling in Pa c-551 and Ht c-552, are utilized to test this correlation between molecular and electronic structure. In conclusion, the structure of the proximal heme pocket of cytochromes c is shown to play a role in determining heme conformation and electronic structure.

Hemes c are characterized by their covalent attachment to a polypeptidevia a widely conserved CXXCH motif. There are multiple biological systems that facilitate hemec biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromesc in Escherichia coli-based expression systems. System III, cytochrome cheme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophiluscytochrome c552 variants was successfully expressed and matured by the CCM system with CXnCH motifs where n = 1–4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1–3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemesc with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.

We demonstrate that Zn(II) porphyrin in Zn(II)cytochrome c (Zn cyt c) is a fluorescence resonance energy transfer (FRET) donor to an Alexa660 dye acceptor. The energy transfer efficiency is dependent on the distance between the two fluorophores as shown through protein denaturation studies of five Zn cyt c variants labeled with Alexa660 in different positions. The relative quantum yield, excitation and emission energies, and labeling efficiencies of this donor−acceptor pair allow for a method of analysis based on sensitized emission of the acceptor. These studies show that Zn(II) porphyrin is an effective energy donor for measurement of molecular-scale distances by FRET.

Out-of-plane (OOP) deformations of the heme cofactor are found in numerous heme-containing proteins and the type of deformation tends to be conserved within functionally related classes of heme proteins. We demonstrate correlations between the heme ruffling OOP deformation and the 13C and 1H nuclear magnetic resonance (NMR) hyperfine shifts of heme aided by density functional theory (DFT) calculations. The degree of ruffling in the heme cofactor of Hydrogenobacter thermophilus cytochrome c552 has been modified by a single amino acid mutation in the second coordination sphere of the cofactor. The 13C and 1H resonances of the cofactor have been assigned using one- and two-dimensional NMR spectroscopy aided by selective 13C-enrichment of the heme. DFT has been used to predict the NMR hyperfine shifts and electron paramagnetic resonance (EPR) g-tensor at several points along the ruffling deformation coordinate. The DFT-predicted NMR and EPR parameters agree with the experimental observations, confirming that an accurate theoretical model of the electronic structure and its response to ruffling has been established. As the degree of ruffling increases, the heme methyl 1H resonances move upfield while the heme methyl and meso 13C resonances move downfield. These changes are a consequence of altered overlap of the Fe 3d and porphyrin π orbitals, which destabilizes all three occupied Fe 3d-based molecular orbitals and decreases the positive and negative spin density on the β-pyrrole and meso carbons, respectively. Consequently, the heme ruffling deformation decreases the electronic coupling of the cofactor with external redox partners and lowers the reduction potential of heme.

The naturally occurring G41S mutation to human (Hs) cytochrome (cyt) c enhances apoptotic activity based upon previous in vitro and in vivo studies, but the molecular mechanism underlying this enhancement remains unknown. Here, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations have been used to identify the structural and electronic differences between wild-type (WT) and G41S Hs cyt c. S41 is part of the hydrogen bonding network for propionate 7 of heme pyrrole ring A in the X-ray structure of G41S Hs cyt c and, compared to WT, G41S Hs cyt c has increased spin density on pyrrole ring C and a faster electron self-exchange rate. DFT calculations illustrate an electronic mechanism where structural changes near ring A can result in electronic changes at ring C. Since ring C is part of the solvent-exposed protein surface, we propose that this heme electronic structure change may ultimately be responsible for the enhanced proapoptotic activity of G41S Hs cyt c.

The electron-donating properties of the axial His ligand to heme iron in cytochromes c (cyts c) are found to be correlated with the midpoint reduction potential (Em) in variants of Hydrogenobacter thermophilus cytochrome c552 (Ht cyt c552) in which mutations have been made in and near the Cys-X-X-Cys-His (CXXCH) heme-binding motif. To probe the strength of the His-Fe(III) interaction, we have measured 13C nuclear magnetic resonance (NMR) chemical shifts for 13CN− bound to heme iron trans to the axial His in Ht Fe(III) cyt c552 variants. We observe a linear relationship between these 13C chemical shifts and Em, indicating that the His-Fe(III) bond strength correlates with Em. To probe a conserved hydrogen bonding interaction between the axial His Hδ1 and the backbone carbonyl of a Pro residue, we measured the pKa of the axial His Hδ1 proton (pKa(2)), which we propose to relate to the His-Fe(III) interaction, reduction potential, and local electrostatic effects. The observed linear relationship between the axial His 13Cβ chemical shift and Em is proposed to reflect histidinate (anionic) character of the ligand. A linear relationship also is seen between the average heme methyl 1H chemical shift and Em which may reflect variation in axial His electron-donating properties or in the ruffling distortion of the heme plane. In summary, chemical shifts of the axial His and exogenous CN− bound trans to His are shown to be sensitive probes of the His-Fe(III) interaction in variants of Ht cyt c552 and display trends that correlate with Em.

FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function.

The heme group in paramagnetic (S = 1/2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole β substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c 552 from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the β substituents on all four heme pyrroles. Here, determination of the orientation of the magnetic axes and analysis of NMR line shapes for H. thermophilus ferricytochrome c 552 is performed. These data reveal that the unusual electronic structure for this protein is a result of fluxionality of the heme axial methionine. It is proposed that the ligand undergoes inversion at the pyramidal sulfur, and the rapid interconversion between two diastereomeric forms results in the unusual heme electronic structure. Thus a fluxional process for a metal-bound amino acid side chain has now been identified.

Horse ferricytochrome c (cyt c) undergoes exchange of one of its axial heme ligands (Met-80) for one or more non-native ligands under denaturing conditions. We have used 1H NMR spectroscopy to detect two conformations of paramagnetic cyt c with non-native heme ligation through a range of urea concentrations. One non-native form is an equilibrium unfolding intermediate observed under partially denaturing conditions and is attributed to replacement of Met-80 with one or more Lys side chains. The second non-native form, in which the native Met ligand is replaced by a His, is observed under strongly denaturing conditions. Thermodynamic analysis of these data indicates a relatively small ΔG (17 kJ/mol) for the transition from native to the Lys-ligated intermediate and a significantly larger ΔG (47 kJ/mol) for the transition from native to the His-ligated species. Although CD and fluorescence data indicate that the equilibrium unfolding of cyt c is a two-state process, these NMR results implicate an intermediate with His-Lys ligation.

It is shown that cytochrome c heme lyase (CCHL) attaches heme covalently to peptides composed of the N-terminal segment of cyt c fused to a non-heme containing protein, lending insight into the substrate specificity of CCHL and providing a new route to artificial heme proteins.