tailieunhanh - Báo cáo khoa học: NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis

The cytochromecd1nitrite reductase fromParacoccus pantotrophuscatalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is thed1 heme, which is synthesized under anaer-obic conditions by usingnirECFD-LGHJNgene products. | ễFEBS Journal NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis Shilpa Bali1 Martin J. Warren2 and Stuart J. Ferguson1 1 Department of Biochemistry University of Oxford UK 2 Department of Biosciences University of Kent Canterbury UK Keywords cytochrome cd1 d1 heme biosynthesis denitrification nitrite reductase Paracoccus pantotrophus tetrapyrrole Correspondence S. J. Ferguson Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU UK Fax 44 1865 613201 Tel 44 1865 613299 E-mail Received 24 June 2010 revised 27 August 2010 accepted 1 October 2010 doi The cytochrome cd1 nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d1 heme which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain DnirF showed that this gene is essential for cd1 assembly and consequently for denitrification which was restored when the DnirF strain was complemented with wild-type plasmid-borne nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and hence must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d1 heme. It is concluded that His41 of NirF which aligns with His200 of the d1 heme domain of cd1 is essential both for this binding and for the production of d1 heme replacement of His41 by Ala Cys Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed. Introduction Denitrification is a four-step transformation of nitrate to dinitrogen gas by various species of .

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