No abstract

Several small molecules and ions, notably carbon monoxide, cyanide, cyanate, and hydrogen sulfide, are potent inhibitors of Ni-containing carbon monoxide dehydrogenases (Ni-CODH) that catalyze very rapid, efficient redox interconversions of CO2 and CO. Protein film electrochemistry, which probes the dependence of steady-state catalytic rate over a wide potential range, reveals how these inhibitors target particular oxidation levels of Ni-CODH relating to intermediates (Cox, Cred1, and Cred2) that have been established for the active site. The following properties are thus established: (1) CO suppresses CO2 reduction (CO is a product inhibitor), but its binding affinity decreases as the potential becomes more negative. (2) Cyanide totally inhibits CO oxidation, but its effect on CO2 reduction is limited to a narrow potential region (between −0.5 and −0.6 V), below which CO2 reduction activity is restored. (3) Cyanate is a strong inhibitor of CO2 reduction but inhibits CO oxidation only within a narrow potential range just above the CO2/CO thermodynamic potential—EPR spectra confirm that cyanate binds selectively to Cred2. (4) Hydrogen sulfide (H2S/HS−) inhibits CO oxidation but not CO2 reduction—the complex on/off characteristics are consistent with it binding at the same oxidation level as Cox and forming a modified version of this inactive state rather than reacting directly with Cred1. The results provide a new perspective on the properties of different catalytic intermediates of Ni-CODH—uniting and clarifying many previous investigations.

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., Biochemistry 2007, 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.

Acetyl-CoA synthase (ACS) is a key enzyme in the Wood–Ljungdahl pathway of anaerobic CO2 fixation, which has long been proposed to operate by a novel mechanism involving a series of protein-bound organometallic (Ni–CO, methyl–Ni, and acetyl–Ni) intermediates. Here we report the first direct structural evidence of the proposed metal–carbon bond. We describe the preparation of the highly active metal-replete enzyme and near-quantitative generation of the kinetically competent carbonylated intermediate. This advance has allowed a combination of Ni and Fe K-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure experiments along with density functional theory calculations. The data reveal that CO binds to the proximal Ni of the six-metal metallocenter at the active site and undergoes dramatic structural and electronic perturbation in forming this organometallic Ni–CO intermediate. This direct identification of a Ni–carbon bond in the catalytically competent CO-bound form of the A cluster of ACS provides definitive experimental structural evidence supporting the proposed organometallic mechanism of anaerobic acetyl-CoA synthesis.

Selective, visible-light-driven conversion of CO2 to CO with a turnover frequency of 20 s−1 under visible light irradiation at 25 °C is catalyzed by an aqueous colloidal system comprising a pseudoternary complex formed among carbon monoxide dehydrogenase (CODH), silver nanoclusters stabilized by polymethacrylic acid (AgNCs-PMAA), and TiO2 nanoparticles. The photocatalytic assembly, which is stable over several hours and for at least 250000 turnovers of the enzyme’s active site, was investigated by separate electrochemical (dark) and fluorescence measurements to establish specific connectivities among the components. The data show (a) that a coating of AgNCs-PMAA on TiO2 greatly enhances its ability as an electrode for CODH- based electrocatalysis of CO2 reduction and (b) that the individual Ag nanoclusters interact directly and dynamically with the enzyme surface, most likely at exposed cysteine thiols. The results lead to a model for photocatalysis in which the AgNCs act as photosensitizers, CODH captures the excited electrons for catalysis, and TiO2 mediates hole transfer from the AgNC valence band to sacrificial electron donors. The results greatly increase the benchmark for reversible CO2 reduction under ambient conditions and demonstrate that, with such efficient catalysts, the limiting factor is the supply of photogenerated electrons.