Hydrogenase spectroscopy

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

The K-edge spectra of [Ni(287)2]2 and [Ni(cdt)2]2 are remarkably similar to each other and to those of natural hydrogenases.196 Some complexes with ligand (289) (R = NMe2, R = H, Me, NMe2) have been characterized using electronic and near infrared spectroscopy.823 Complex [Ni(289)2]2 served to study phase transformation behavior by microscopy and DSC.824... 

Electrochemical cell for measurement of redox potentials of hydrogenase by EPR spectroscopy... 

As will be explained in Chapter 7, spectroscopic methods are a powerful way to probe the active sites of the hydrogenases. Often spectroscopic methods are greatly enhanced by judicious enrichment of the active sites with a stable isotope. For example, Mossbauer spectroscopy detects only the isotope Fe, which is present at only 2.2 per cent abundance in natural iron. Hydrogen atoms, which cannot be seen by X-ray diffraction for example, can be studied by EPR and ENDOR spectroscopy, which exploit the hyperfine interactions between the unpaired electron spin and nuclear spins. More detailed information has been derived from hyperfine interactions with nuclei such as Ni and Se, in the active sites. In FTTR spec-... 

Figure 5.10 Redox titration of the Ni-C EPR signal in D. gigas hydrogenase, in the presence of mediators under partial pressure of H2. (A) Titration monitored by EPR spectroscopy (data from Cammack et al. 1982, 1987).The data points were obtained by removing samples from a vessel as shown in Fig. 5.8. Data NiA signal A NiC signal. (B) Titration monitored by FTIR spectroscopy (data from De Lacey et al. 1997).The spectra were recorded directly in a sealed optically transparent thin-layer electrode cell. Note that the oxidized and reduced species, which are undetectable by EPR, can be measured. Data o I946cm (NiB state) 1914+ 1934cm (NiSR state) A 1952cm (NiA state) 1940cm (NiR state).

Atomic absorption spectrometry, EPR spectroscopy and inductively coupled plasma (ICP) analysis had shown that the D. gigas hydrogenase contains one nickel and twelve ( 1) iron atoms, eleven of which are distributed among the three [FeS] clusters. This strongly suggested that the remaining twelfth iron atom could be one of the two metal ions revealed by the active site electron density. To verify this... 

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. 

D. gigas hydrogenase can be considered a case study for the application of Mossbauer and EPR spectroscopies in conjunction with redox titration methodologies. H2 (the substrate/product of the reaction) was used to control the redox state of the enzyme by varying the partial pressure of the gas. By doing so, several samples of the enzyme were obtained in different oxidation states and investigated in parallel both by Mossbauer and EPR spectroscopies. 

As described above, the combination of EPR and Mossbauer spectroscopies, when applied to carefully prepared parallel samples, enables a detailed characterization of all the redox states of the clusters present in the enzyme. Once the characteristic spectroscopic properties of each cluster are identified, the determination of their midpoint redox potentials is an easy task. Plots of relative amounts of each species (or some characteristic intensive property) as a function of the potential can be fitted to Nernst equations. In the case of the D. gigas hydrogenase it was determined that those midpoint redox potentials (at pFi 7.0) were —70 mV for the [3Fe-4S] [3Fe-4S]° and —290 and —340mV for each of the [4Fe-4S]> [4Fe-4S] transitions. 

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). 

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