EPR AND MOSSBAUER SPECTROSCOPIC STUDIES ON THE TETRAMERIC, NAD-LINKED HYDROGENASE OF NOCARDIA-OPACA-1B AND ITS 2 DIMERS .1. THE BETA-DELTA-DIMER - A PROTOTYPE OF A SIMPLE HYDROGENASE

The cytoplasmic, tetrameric NAD-linked hydrogenase from Nocardia opaca Ib can be separated in two dimeric substructures, an ay-dimer with NADH:electron acceptor oxidoreductase (diaphorase) activity and a PG-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 PS-dimer contained, as analyzed by inductively coupled plasma mass spectrometry (TCP-MS), 3.5-3.9 iron atoms and 1.3-1.7 nickel atoms per dimer molecule, EPR and Mossbauer 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 [4Fe-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 PG-dimer were detectable in the oxidized as well as in the reduced state, The diaphorase-active ay-dimer was free of nickel and the iron content corresponded to 11.2-12.8 Fe atoms per dimer molecule, From EPR and Mossbauer 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 diner 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.