Hydrogenases classes

For a better understanding of the design principles of the native enzymes, a comparison of the two major hydrogenase classes is useful. 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

Especially for this latter class of hydrogenases, great effort has been devoted to the purification and the characterization of the metal centers involved, using biochemical, genetic, spectroscopic (IR, EPR, Mossbauer, MCD, EXAFS, and mass spectrometry), and crystallographic techniques 152, 165, 166). 

A different mechanism for reduction processes by [Fe]-hydrogenase 56 is assumed. The hydride generated by splitting dihydrogen is directly transferred to an electrophilic organic center in methenyltetrahydrocyanopterin. As no electrons need to be transferred this reaction requires only one metal center. Due to its structure the center of [Fe]-hydrogenase 56 does not count to the class of ferrates. 

Nickel is found in thiolate/sulflde environment in the [NiFe]-hydrogenases and in CODH/ACS.33 In addition, either a mononuclear Ni-thiolate site or a dinuclear cysteine-S bridged structure are assumed plausible for the new class of Ni-containing superoxide dismutases, NiSOD (A).34 [NiFe]-hydrogenase catalyzes the two-electron redox chemistry of dihydrogen. Several crystal structures of [NiFe]-hydrogenases have demonstrated that the active site of the enzyme consists of a heterodinuclear Ni—Fe unit bound to thiolate sulfurs of cysteine residues with a Ni—Fe distance below 3 A (4) 35-39 This heterodinuclear active site has been the target of extensive model studies, which are summarized in Section 6.3.4.12.5. 

Nakamura et al. 1999 Takahashi and Nakamura 1999) may be required for assembly and insertion of the [Fe-S] clusters in several different classes of hydrogenases although there is no direct evidence for this. 

Hydrogenase catalyses the simplest redox-linked chemical reaction in Nature, so one might assume that the task of solving the puzzle of how hydrogenases actually do this is a simple one. As the chapters in this book describe, the scientists involved did not anticipate the peculiarities discovered in these ancient enzymes. As already described in the previous chapters, we now know of two classes of enzymes which, when in the pure state, can activate H2 without added cofactors. 

In conclusion, the presented dinuclear iron structure is the first example of a bio-mimetic iron compound, which can be regarded as a first generation model for the class of [Fe]-only hydrogenases. The complex incorporates both relevant carbon monoxide ligands, as well as three bridging thiolato ligands, which could be possibly present in the active site of these enzymes. 

Berber, Y., Fauque, G. D., LeGall, J., Choi, E. S., Peck, H. D. Jr and Lespinat, P. A. (1987) Inhibition studies of three classes of Desulfovibrio hydrogenase Application to the further characterization of the multiple hydrogenases found in Desulfovibrio vulgaris Hildenborough. Biochem. Biophys. Res. Commun., 146, 147-53. 

Paschos, A, Class, R. S. and Bock, A. (2001) Carbamoylphosphate requirement for synthesis of the active center of [NiFe]-hydrogenases. FEBS Lett., 488, 9-12. 

Fauque G, Peck HD Jr, Moura JJG, et al. 1988. The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio. FEMS Microbiol Rev 54 299-344. 

Both hydrogenases and carbon monoxide oxidoreductases contain iron-sulfur clusters in addition to nickel. It may be noted that in addition to the Ni hydrogenases, there is another class of Fe hydrogenases, such as those in clostridia, which contain no nickel but have a specialized type of iron-sulfur cluster (28a, 28b). Therefore, it has to be established that the nickel in Ni hydrogenases is the active site as will be seen later, there is a considerable amount of circumstantial evidence for this. 

The soluble hydrogenase from the hydrogen-oxidizing bacterium N. opaca is one of a class of hydrogenases that contain flavin and use nicotinamide adenine dinucleotide (NAD) as electron acceptor. The protein consists of four dissimilar subunits and contains approximately four atoms of nickel, one FMN, three [Fe-4S] clusters, one [2Fe-2S] cluster, and up to one [3Fe-xS] cluster (82). Two of the nickel atoms were readily removed by dialysis, in contrast to the nickel in most hydrogenases. The enzyme would only catalyze electron transfer from hydrogen to NAD if cations, of which Ni2+ is the most effective, were added. In the absence of the cations, the enzyme could be separated as... 

Proteins and antibodies are natural substrates for affinity columns because of the nature of the enzyme recognition site and the antibody-antigen interaction sites. They have a three-dimensional shape and electrical charge distributions that interact with only specific molecules or types of molecules. Once these substrate sites are identified, molecules can be isolated or synthesized with the key characteristics and used to build affinity supports. These substrates are often bound to a 6-carbon spacer so that they protrude farther away from the packing surface toward the mobile phase and are therefore more available. Certain natural and synthetic dyes have been found to serve as substrate mimics for a class of enzymes call hydrogenases and have been used to build affinity columns for their purification. 

These features demonstrate how important it is to study and understand hydrogenase structure and function when designing biomimetic (or bioinspired) systems for efficient hydrogen generation or utilization. This section will give insight into this class of enzymes. 

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