EPR and Mössbauer spectroscopic studies on the tetrameric, NAD-linked hydrogenase of Nocardia opaca 1b and its two dimers: 1. The βδ-dimer-a prototype of a simple hydrogenase

The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca 1b can be separated in two dimeric substructures, an αγ-dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a βδ-dimer which displays hydrogenase activity with artificial electron carriers. These two dimers were preparatively isolated by a FPLC Mono Q procedure in the absence of nickel and at alkaline pH values. The hydrogenase-active βδ-dimer contained, as analyzed by inductively coupled plasma mass spectrometry (ICP-MS), 3.5-3.9 iron atoms and 1.3-1.7 nickel atoms per dimer molecule. EPR and Mössbauer spectra indicated the presence of a [4Fe-4S] cluster. This center turned out to be extremely labile towards oxidants. Oxidation led to irreversible convertion into a [3Fe-4S] form, thus representing an artifact and not a regulatory state of the cluster. The midpoint redox potential of the [4Fe-4S] cluster was determined to be -385 mV. Very weak EPR Ni signals of the βδ-dimer were detectable in the oxidized as well as in the reduced state. The diaphorase-active αγ-dimer was free of nickel and the iron content corresponded to 11.2-12.8 Fe atoms per dimer molecule. From EPR and Mössbauer measurements it was concluded that this dimer contained two [4Fe-4S] clusters, one [2Fe-2S] and one [3Fe-4S] cluster. In accordance with the results obtained for the dimer proteins, for the whole enzyme an iron content of 15.8-16.2 atoms per enzyme molecule have been determined. EPR spectra and spectrum simulations of the native hydrogenase corroborate the cluster assignments of the two dimers: in total the enzyme contains one [2Fe-2S] cluster, one [3Fe-4S] cluster and three [4Fe-4S] clusters.