tailieunhanh - Báo cáo khoa học: The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774 Re-evaluation of the spectroscopic data and redox properties

The cytochromec nitrite reductase is isolated from the membranes of the sulfate-reducing bacteriumDesulfovibrio desulfuricansATCC 27774 as a heterooligomeric complex composedby two subunits (61 kDa and19 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 sub-units from one another is accomplished by gel filtration chromatography in the presence of SDS. | Eur. J. Biochem. 270 3904-3915 2003 FEBS 2003 doi The isolation and characterization of cytochrome c nitrite reductase subunits NrfA and NrfH from Desulfovibrio desulfuricans ATCC 27774 Re-evaluation of the spectroscopic data and redox properties Maria Gabriela Almeida1 Sofia Madeira2. Luisa L Goncalves1 Robert Huber2 Carlos A Cunha1 . . Maria Joao Romao1 Cristina Costa1 Jorge Lampreia1 Jose J. G. Moura1 and Isabel Moura1 1REQUIMTE CQFB Departamento de Quimica Faculdade de Ciencias e Tecnologia Universidade Nova de Lisboa Portugal 2Max-Planck-Institut fur Biochemie Abt. Strukturforschung Martinsried Germany 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 EPR and Mossbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure Cunha . Macieira S. Dias . Almeida . Goncalves . Costa C. Lampreia J. Huber R. Moura . Moura I. Romao M. 2003 J. Biol. Chem. 278 1745517465 we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site -80 mV as well as a quite unusual high reduction potential 150 mV .