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The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro , with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

While a general model of H2 activation has been proposed for [FeFe]-hydrogenases, the structural and biophysical properties of the intermediates of the H-cluster catalytic site have not yet been discretely defined. Electron paramagnetic resonance (EPR) spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the H-cluster catalytic site, a [4Fe-4S]H subcluster linked by a cysteine thiolate to an organometallic diiron subsite with CO, CN, and dithiolate ligands, in [FeFe]-hydrogenase HydA1 from Chlamydomonas reinhardtii (CrHydA1). Oxidized CrHydA1 displayed a rhombic 2.1 EPR signal (g = 2.100, 2.039, 1.997) and an FTIR spectrum previously assigned to the oxidized H-cluster (Hox). Reduction of the Hox sample with 100% H2 or sodium dithionite (NaDT) nearly eliminated the 2.1 signal, which coincided with appearance of a broad 2.3-2.07 signal (g = 2.3-2.07, 1.863) and/or a rhombic 2.08 signal (g = 2.077, 1.935, 1.880). Both signals displayed relaxation properties similar to those of [4Fe-4S] clusters and are consistent with an S = 1/2 H-cluster containing a [4Fe-4S]H(+) subcluster. These EPR signals were correlated with differences in the CO and CN ligand modes in the FTIR spectra of H2- and NaDT-reduced samples compared with Hox. The results indicate that reduction of [4Fe-4S]H from the 2+ state to the 1+ state occurs during both catalytic H2 activation and proton reduction and is accompanied by structural rearrangements of the diiron subsite CO/CN ligand field. Changes in the [4Fe-4S]H oxidation state occur in electron exchange with the diiron subsite during catalysis and mediate electron transfer with either external carriers or accessory FeS clusters.