NirF is a periplasmic protein that binds d1 heme as part of its essential role in d1 heme biogenesis

The cytochrome cd(1) 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 d(1) heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, Delta nirF, showed that this gene is essential for cd(1) assembly and consequently for denitrification, which was restored when the Delta nirF 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 d(1) heme. It is concluded that His41 of NirF, which aligns with His200 of the d(1) heme domain of cd(1), is essential both for this binding and for the production of d(1) heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed.