Shewanella oneidensis MR-1 respires CdSe quantum dots for photocatalytic hydrogen evolution.

Living bio-nano systems for artificial photosynthesis are of growing interest. Typically, these systems use photoinduced charge transfer to provide electrons for microbial metabolic processes, yielding a biosynthetic solar fuel. Here, we demonstrate an entirely different approach to constructing a living bio-nano system, in which electrogenic bacteria respire semiconductor nanoparticles to support nanoparticle photocatalysis. Semiconductor nanocrystals are highly active and robust photocatalysts for hydrogen (H2) evolution, but their use is hindered by the oxidative side of the reaction. In this system, Shewanella oneidensis MR-1 provides electrons to a CdSe nanocrystalline photocatalyst, enabling visible light-driven H2 production. Unlike microbial electrolysis cells, this system requires no external potential. Illuminating this system at 530 nm yields continuous H2 generation for 168 h, which can be lengthened further by replenishing bacterial nutrients.

The two redox states of the human NEET proteins' [2Fe-2S] clusters.

The NEET proteins constitute a unique class of [2Fe-2S] proteins. The metal ions bind to three cysteines and one histidine. The proteins' clusters exist in two redox states; the oxidized protein (containing two FeIII ions) can transfer the cluster to apo-acceptor protein(s), while the reduced form (containing one ferrous ion) remains bound to the protein frame. Here, we perform in silico and in vitro studies on human NEET proteins in both reduced and oxidized forms. Quantum chemical calculations on all available human NEET proteins structures suggest that reducing the cluster weakens the Fe-NHis and Fe-SCys bonds, similar to what is seen in other Fe-S proteins (e.g., ferredoxin and Rieske protein). We further show that the extra electron in the [2Fe-2S]+ clusters of one of the NEET proteins (mNT) is localized on the His-bound iron ion, consistently with our previous spectroscopic studies. Kinetic measurements demonstrate that the mNT [2Fe-2S]+ is released only by an increase in temperature. Thus, the reduced state of human NEET proteins [2Fe-2S] cluster is kinetically inert. This previously unrecognized kinetic inertness of the reduced state, along with the reactivity of the oxidized state, is unique across all [2Fe-2S] proteins. Finally, using a coevolutionary analysis, along with molecular dynamics simulations, we provide insight on the observed allostery between the loop L2 and the cluster region. Specifically, we show that W75, R76, K78, K79, F82 and G85 in the latter region share similar allosteric characteristics in both redox states.

Semiconductor nanocrystal photocatalysis for the production of solar fuels.

Colloidal semiconducting nanocrystals (NCs) are powerful elements of a photocatalytic system useful for enabling a variety of chemical transformations owing to their strong light-absorbing properties and high degree of size-, shape-, and composition-tunability. Key to their utility is our understanding of the photoinduced charge transfer processes required for these photochemical transformations. This Perspective will focus on the implementation of semiconductor NCs for photochemical fuel formation. Three general system designs for photocatalytic proton reduction using semiconductor NCs will be reviewed: metal-semiconductor heterostructures, NC photosensitizers with molecular catalysts, and hydrogenase-based systems. Other relevant reactions toward solar fuel targets, such as CO2 and N2 reductions with NCs, will also be highlighted. Illustrating the versatile roles that NCs can play in light-driven chemical reactions, advances made toward NC-catalyzed organic transformations will be discussed. Finally, we will share a few concluding thoughts and perspectives on the future of the field, with a focus on goals toward improving and implementing NC-based technologies for solar fuel development.

Tuning Mechanism through Buffer Dependence of Hydrogen Evolution Catalyzed by a Cobalt Mini-enzyme.

Cobalt-mimochrome VI*a (CoMC6*a) is a synthetic mini-protein that catalyzes aqueous proton reduction to hydrogen (H2). In buffered water, there are multiple possible proton donors, complicating the elucidation of the mechanism. We have found that the buffer pKa and sterics have significant effects on activity, evaluated via cyclic voltammetry (CV). Protonated buffer is proposed to act as the primary proton donor to the catalyst, specifically through the protonated amine of the buffers that were tested. At a constant pH of 6.5, catalytic H2 evolution in the presence of buffer acids with pKa values ranging from 5.8 to 11.6 was investigated, giving rise to a potential-pKa relationship that can be divided into two regions. For acids with pKa values of ≤8.7, the half-wave catalytic potential (Eh) changes as a function of pKa with a slope of -128 mV/pKa unit, and for acids with pKa of ≥8.7, Eh changes as a function of pKa with a slope of -39 mV/pKa unit. In addition, a series of buffer acids were synthesized to explore the influence of steric bulk around the acidic proton on catalysis. The catalytic current in CV shows a significant decrease in the presence of the sterically hindered buffer acids compared to those of their parent compounds, also consistent with the added buffer acid acting as the primary proton donor to the catalyst and showing that acid structure in addition to pKa impacts activity. These results demonstrate that buffer acidity and structure are important considerations when optimizing and evaluating systems for proton-dependent catalysis in water.

Buffer pKa Impacts the Mechanism of Hydrogen Evolution Catalyzed by a Cobalt Porphyrin-Peptide.

The effect of buffer pKa on the mechanism of electrochemical hydrogen evolution catalyzed by a cobalt porphyrin peptide (CoMP11-Ac) at constant pH is presented. The addition of buffer to CoMP11-Ac in water and KCl leads to an enhancement of the catalytic current of up to 200-fold relative to its value in the absence of a buffer. Two distinct catalytic regimes are identified as a function of the buffer pKa. In the presence of buffers with pKa ≤ 7.4, a fast catalysis regime limited by diffusion of buffer is reached. The catalytic half-wave potential (Eh) shifts anodically (from -1.42 to -1.26 V vs Ag/AgCl/KCl(1M)) as the buffer pKa decreases from 7.4 to 5.6, proposed to result from fast Co(III)-H formation following the catalysis-initiating Co(II/I) reduction. With higher-pKa buffers (pKa > 7.7), an Eh = -1.42 V, proposed to reflect the Co(II/I) couple, is maintained independent of the buffer pKa, consistent with rate-limiting Co(III)-H formation under these conditions. We conclude that the buffer species pKa impacts catalytic current and potential and the rate-determining step of the reaction.

Light-driven catalysis with engineered enzymes and biomimetic systems.

Efforts to drive catalytic reactions with light, inspired by natural processes like photosynthesis, have a long history and have seen significant recent growth. Successfully engineering systems using biomolecular and bioinspired catalysts to carry out light-driven chemical reactions capitalizes on advantages offered from the fields of biocatalysis and photocatalysis. In particular, driving reactions under mild conditions and in water, in which enzymes are operative, using sunlight as a renewable energy source yield environmentally friendly systems. Furthermore, using enzymes and bioinspired systems can take advantage of the high efficiency and specificity of biocatalysts. There are many challenges to overcome to fully capitalize on the potential of light-driven biocatalysis. In this mini-review, we discuss examples of enzymes and engineered biomolecular catalysts that are activated via electron transfer from a photosensitizer in a photocatalytic system. We place an emphasis on selected forefront chemical reactions of high interest, including CH oxidation, proton reduction, water oxidation, CO2 reduction, and N2 reduction.

Photochemical Hydrogen Evolution from Neutral Water with a Cobalt Metallopeptide Catalyst.

CoGGH, a Gly-Gly-His tripeptide coordinated to a cobalt ion, is shown to catalyze the reduction of aqueous protons to hydrogen (H2) in a light-driven reaction in water near neutral pH. Using [Ru(bpy)3]2+ as a photosensitizer and ascorbate as an electron donor, a turnover number up to 2200 with respect to CoGGH has been observed with the system remaining active for more than 48 h. The reaction conditions that favor H2 production are consistent with a reductive quenching mechanism. Results also suggest that CoGGH is robust under these reaction conditions and loss of activity over time results from [Ru(bpy)3]2+ degradation.

Cobalt Metallopeptide Electrocatalyst for the Selective Reduction of Nitrite to Ammonium.

A cobalt-tripeptide complex (CoGGH) is developed as an electrocatalyst for the selective six-electron, eight-proton reduction of nitrite to ammonium in aqueous buffer near neutral pH. The onset potential for nitrite reduction occurs at -0.65 V vs Ag/AgCl (1 M KCl). Controlled potential electrolysis at -0.90 V generates ammonium with a faradaic efficiency of 90 ± 3% and a turnover number of 3550 ± 420 over 5.5 h. CoGGH also catalyzes the reduction of the proposed intermediates nitric oxide and hydroxylamine to ammonium. These results reveal that a simple metallopeptide is an active functional mimic of the complex enzymes cytochrome c nitrite reductase and siroheme-containing nitrite reductase.

Influence of heme c attachment on heme conformation and potential.

Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CX3CH or CX4CH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CX3CH and CX4CH variants of Hydrogenobacter thermophilus cytochrome c552. In addition, a novel CXCH variant is studied. 1H and 13C NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CX4CH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.

Discovery of the magnetic behavior of hemoglobin: A beginning of bioinorganic chemistry.

Two articles published by Pauling and Coryell in PNAS nearly 80 years ago described in detail the magnetic properties of oxy- and deoxyhemoglobin, as well as those of closely related compounds containing hemes. Their measurements revealed a large difference in magnetism between oxygenated and deoxygenated forms of the protein and, along with consideration of the observed diamagnetism of the carbonmonoxy derivative, led to an electronic structural formulation of oxyhemoglobin. The key role of hemoglobin as the main oxygen carrier in mammalian blood had been established earlier, and its allosteric behavior had been described in the 1920s. The Pauling-Coryell articles on hemoglobin represent truly seminal contributions to the field of bioinorganic chemistry because they are the first to make connections between active site electronic structure and the function of a metalloprotein.

Efficient and Flexible Preparation of Biosynthetic Microperoxidases.

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.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c.

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation. In this work, we studied Pseudomonas aeruginosa cytochrome c551 and its F7A mutant. These two proteins, although similar in their X-ray crystal structure, display a significant difference in their heme out-of-plane deformations, mainly along the ruffling coordinate. Resonance Raman and vibrational coherence measurements also indicate significant differences in ruffling-sensitive modes, particularly the low-frequency γa mode found between ∼50-60 cm(-1). This supports previous assignments of γa as having a large ruffling content. Measurement of the photoreduction kinetics finds an order of magnitude decrease of the photoreduction cross-section in the F7A mutant, which has nearly twice the ruffling deformation as the WT. Additional measurements on cytochrome c demonstrate that heme ruffling is correlated exponentially with the electron transfer rates and suggest that ruffling could play an important role in redox control. A major relaxation of heme ruffling in cytochrome c, upon binding to the mitochondrial membrane, is discussed in this context.

Biological significance and applications of heme c proteins and peptides.

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.

Semisynthetic and Biomolecular Hydrogen Evolution Catalysts.

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.

Hydrogen Evolution from Water under Aerobic Conditions Catalyzed by a Cobalt ATCUN Metallopeptide.

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.

Effects of protein structure on iron-polypeptide vibrational dynamic coupling in cytochrome c.

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 (57)Fe-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.

Methionine ligand lability of homologous monoheme cytochromes c.

Direct electrochemical analysis of adsorbed bacterial monoheme cytochromes c has revealed a phenomenological loss of the axial methionine when examined using pyrolytic "edge-plane" graphite (EPG) electrodes. While prior findings have reported that the Met-loss state may be quantitatively understood using the cytochrome c from Hydrogenobacter thermophilus as a model system, here we demonstrate that the formation of the Met-loss state upon EPG electrodes can be observed for a range of cytochrome orthologs. Through an electrochemical comparison of the wild-type proteins from organisms of varying growth temperature optima, we establish that Met-ligand losses at graphite surfaces have similar energetics to the "foldons" for known protein folding pathways. Furthermore, a downward shift in reduction potential to approximately -100 mV vs standard hydrogen electrode was observed, similar to that of the alkaline transition found in mitochondrial cytochromes c. Pourbaix diagrams for the Met-loss forms of each cytochrome, considered here in comparison to mutants where the Met-ligand has been substituted to His or Ala, suggest that the nature of the Met-loss state is distinct from either a His-/aquo- or a bis-His-ligated heme center, yet more closely matches the pKa values found for bis-His-ligated hemes., We find the propensity for adoption of the Met-loss state in bacterial monoheme cytochromes c scales with their overall thermal stability, though not with the specific stability of the Fe-Met bond.

Heme-protein vibrational couplings in cytochrome c provide a dynamic link that connects the heme-iron and the protein surface.

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 (57)Fe-labeled ferric Hydrogenobacter thermophilus cytochrome c(552), including (13)C(8)-heme-, (13)C(5)(15)N-Met-, and (13)C(15)N-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 c(552) 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.

Hydrogen evolution from neutral water under aerobic conditions catalyzed by cobalt microperoxidase-11.

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 × 10(4). Catalyst activity has low sensitivity to oxygen. The results show promise as a hydrogenase functional mimic derived from a biomolecule.

Structural characterization of nitrosomonas europaea cytochrome c-552 variants with marked differences in electronic structure.

Nitrosomonas europaea cytochrome c-552 (Ne c-552) variants with the same His/Met axial ligand set but with different EPR spectra have been characterized structurally, to aid understanding of how molecular structure determines heme electronic structure. Visible light absorption, Raman, and resonance Raman spectroscopy of the protein crystals was performed along with structure determination. The structures solved are those of Ne c-552, which displays a "HALS" (or highly anisotropic low-spin) EPR spectrum, and of the deletion mutant Ne N64Δ, which has a rhombic EPR spectrum. Two X-ray crystal structures of wild-type Ne c-552 are reported; one is of the protein isolated from N. europaea cells (Ne c-552n, 2.35 Å resolution), and the other is of recombinant protein expressed in Escherichia coli (Ne c-552r, 1.63 Å resolution). Ne N64Δ crystallized in two different space groups, and two structures are reported [monoclinic (2.1 Å resolution) and hexagonal (2.3 Å resolution)]. Comparison of the structures of the wild-type and mutant proteins reveals that heme ruffling is increased in the mutant; increased ruffling is predicted to yield a more rhombic EPR spectrum. The 2.35 Å Ne c-552n structure shows 18 molecules in the asymmetric unit; analysis of the structure is consistent with population of more than one axial Met configuration, as seen previously by NMR. Finally, the mutation was shown to yield a more hydrophobic heme pocket and to expel water molecules from near the axial Met. These structures reveal that heme pocket residue 64 plays multiple roles in regulating the axial ligand orientation and the interaction of water with the heme. These results support the hypothesis that more ruffled hemes lead to more rhombic EPR signals in cytochromes c with His/Met axial ligation.