NiFe hydrogenase spectroscopy

The [NiFe] hydrogenase metal sites were completely defined by spectroscopy and X-ray diffraction studies, as discussed before. The... 

Using EPR and Mossbauer spectroscopies to probe the metal clusters in [NiFe] hydrogenase... 

EPR and Mossbauer spectroscopies have been successfully used to characterize iron-sulfur clusters. Hydrogenases are no exception. Here, we will describe the knowledge gained from applying these spectroscopies to the study of [NiFe] hydrogenase. 

We do not know exactly where the hydrogen binds at the active site. We would not expect it to be detectable by X-ray diffraction, even at 0.1 nm resolution. EPR (Van der Zwaan et al. 1985), ENDOR (Fan et al. 1991b) and electron spin-echo envelope modulation (ESEEM) (Chapman et al. 1988) spectroscopy have detected hyperfine interactions with exchangeable hydrous in the NiC state of the [NiFe] hydrogenase, but have not so far located the hydron. It could bind to one or both metal ions, either as a hydride or H2 complex. Transition-metal chemistry provides many examples of hydrides and H2 complexes (see, for example. Bender et al. 1997). These are mostly with higher-mass elements such as osmium or ruthenium, but iron can form them too. In order to stabilize the compounds, carbonyl and phosphine ligands are commonly used (Section 6). 

Formic acid irradiation of, 3 183 reaction with hydrogen atom, 3 191 Formylium cation, 9 231 Formylmethanofuran dehydrogenase, Methano-bacterium wolfei, 40 73 Fossil fuel, radiocarbon and, 3 311-312 Four-coordinated metal centers, 37 19 Fourier-transform infrared spectroscopy, NiFe hydrogenase, 47 295-298, 299, 303 Fourier-transform ion cyclotron resonance... 

As ascribed, the EPR spectrum with g = 2.10 can be low-spin Fec(III). When the isolated enzyme is reductively titrated this signal disappears at a potential Emj -0.3 V [65]. This would seem to indicate that the putative Fec(III) form is not relevant, at least not to hydrogen-production activity. The cubane is a one-electron acceptor as it can shuttle between the 2+ and 1 + oxidation states. Therefore, if the active center were to take up a total of two electrons, then the oxidation state of the Fec would, as least formally, shuttle between II and I. Recently, a redox transition in Fe hydrogenase with an Em below the H2/H+ potential has been observed in direct electrochemistry [89]. This superreduced state has not been studied by spectroscopy. It might well correspond to the formal Fec(I) state. For NiFe hydrogenases Fec(I) has recently been proposed as a key intermediate in the catalytic cycle [90] (cf. Chapter 9). 

FeFe] Hydrogenase Less information on the electronic structure is available from EPR on [FeFe] hydrogenase as compared to [NiFe] hydrogenase since only the Hox state (and Hox-CO) is paramagnetic. In particular, the hydride-carrying species (Hred) cannot be characterized by EPR techniques. However, FTIR and Moss-bauer spectroscopy have revealed important information on the intermediate states of the H cluster [128, 129]. 

Pandelia ME, Ogata H, Lubitz W. Intermediates in the catalytic cycle of [NiFe] hydrogenase functional spectroscopy of the active site. ChemPhysChem. 2010 11(6) 1127-40. 

Kurkin S, George SJ, Thorneley RNF, Albracht SPJ. Hydrogen-induced activation of the [NiFe]-hydrogenase from Allochromatium vinosum as studied by stopped-flow infrared spectroscopy. Biochemistry. 2004 43(21) 6820-31. 

Comparison of EPR [25] and ESEEM [26] spectra of the NiFe-hydrogenases in HjO and D2O indicates the presence of exchangable protons in the vicinity of the nickel in the Ni-C state. Q-band ENDOR spectroscopy [27] indicates two types of exchangable protons. These exchangable protons only interact weakly with nickel and so it is concluded that they are outside the first coordination... 

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