Insights in active sites: Hydrogen‐conversion by hydrogenase is mediated by a sophisticated, metal‐containing catalytic center. Resonance Raman spectroscopy is used for the first time in the characterization of the active site of these biocatalysts. An integrated spectroscopic and computational approach gives insights into structural and photochemical properties of the active site of an oxygen‐tolerant [NiFe] hydrogenase.

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global (13)C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE-->ZZZ), which involves only small protein structural changes.

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Surface-enhanced infrared absorption spectroscopy is used in situ to determine the electrochemical stability of organic interfaces deposited onto the surface of nanostructured, thin-film gold electrodes via the electrochemical reduction of diazonium salts. These interfaces are shown to exhibit a wide electrochemical stability window in both acetonitrile and phosphate buffer, far surpassing the stability window of thiol-derived self-assembled monolayers. Using the same in situ technique, the application of radical scavengers during the electrochemical reduction of diazonium salts is shown to moderate interface formation. Consequently, the heterogeneous charge-transfer resistance can be reduced sufficiently to enhance the direct electron transfer between an immobilized redox-active enzyme and the electrode. This was demonstrated for the oxygen-tolerant [NiFe] hydrogenase from the "Knallgas" bacterium Ralstonia eutropha by relating its electrochemical activity for hydrogen oxidation to the interface properties.

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.