Hydrogenase determination

The hydrogenase-like proteins are known to catalyze the H/D exchange reaction, typical of hydrogenase (58). It remains to be determined to what extent the hydrogen binding site of these proteins re-... 

The first Ni Mossbauer spectrum of nickel in a bioinorganic compound with determinable EFG and isomer shift was reported for a nickel complex compound with planar [NiSJ core and considered as a model compound for hydrogenase. This Mossbauer spectrum from the formal Ni compound is presented in Fig. 7.16. The observed quadrupolar interaction can be understood in terms of ligand field theory. In this approach, the b g and levels (d y2 and d ) are not occupied which is expected to cause a large negative EFG contribution [32]. 

The in vitro hydrogenase activity was determined by using a gas chromatograph (Hewlett Packard 5890 A Series II, column molecular Sieve 5 A, Mesh 60/80). As described before [Happe and Naber, 1993], methylviologen reduced by sodium dithionite was used as electron donor. One unit is defined as the amount of hydrogenase evolving 1 pmol Hi-min1 at 25°C. 

Activity of the various states of the [NiFe] hydrogenase from A. vinosum as determined with a Pt electrode at 30°C... 

Figure SJ Activity of the various states of the [NiFe] hydrogenase from A. vinosum as determined with a Pt electrode at 30°C.The reaction was performed in SOmM Tris/HCI (pH 8.0) in a volume of 2 ml. Oxygen was scavenged by adding glucose (90 mM) and glucose oxidase (2.5 mg/ml). Hydrogen peroxide was removed by catalase. When the system was anaerobic, an aliquot of H2-saturated water was added, and a little later enzyme (S-IOnM) was injected. Benzyl viologen (4.2mM) was used as electron acceptor.

Let us, for example, determine the of a [4Fe-4S] cluster in hydrogenase. The cluster has an EPR signal in its reduced form so we must rewrite the Nernst equation as... 

Many essential strnctural and functional features of hydrogenases have been derived from a wealth of various biochemical and spectroscopic methods. However, the knowledge of their atomic architectures have been obtained only very recently with the determination of the crystal structures of several hydrogenases belonging to both [NiFe] and [Fe] families (Table 6.1). These results have given a firm and nniqne strnctural basis to understand how these enzymes are actually working. 

Two [Fe] hydrogenase structures have so far been determined from C. pasteurianum (Cp) (Peters et al. 1998) and D. desulfuricans (Dd) (Nicolet et al. 1999). They have in common a large domain, which contains the catalytic site and three [4Fe-4S] iron sulfur clusters. The catalytic site and the closest (proximal) cluster are deeply buried inside the protein between two domains (or lobes), with access to a third, ferredoxin-like, domain that contains the two remaining (medial and distal) clusters. By contrast with [NiFe] hydrogenases the proximal [4Fe-4S] cluster is directly bridged to the bin-uclear active site by a cysteic thiolate (Fig. 6.12). 

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. 

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