The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774

Abstract

The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH,respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lackof knowledge on the heme content and the oligomeric enzyme status. Based on EPRandMo¨ ssbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Gonçalves, L.M.L., Costa, C., Lampreia, J.,Huber,R., Moura, J.J.G., Moura, I. & Romão, M. (2003) J. Biol. Chem. 278, 17455– 17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes.NrfAcontains the high-spin catalytic site ()80mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of theNrfH subunit. The four NrfH hemes are all in a low-spin state (S ¼ 1/2).One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential.