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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, ΔnirF, showed that this gene is essential for cd₁ assembly and consequently for denitrification, which was restored when the ΔnirF 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.

Original publication




Journal article



Publication Date





4944 - 4955


Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Cytochromes, DNA, Bacterial, Gene Deletion, Genes, Bacterial, Genetic Complementation Test, Heme, Molecular Sequence Data, Nitrite Reductases, Operon, Oxidoreductases, Paracoccus pantotrophus, Periplasmic Proteins, Protein Processing, Post-Translational, Protein Sorting Signals, Recombinant Proteins, Sequence Deletion, Sequence Homology, Amino Acid