NirF is a periplasmic protein that binds d₁ heme as part of its essential role in d₁ heme biogenesis.
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.
Department of Biochemistry, University of Oxford, UK.
Shilpa
S
Bali
Martin J
MJ
Warren
Stuart J
SJ
Ferguson
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006418:Heme / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009876:Operon; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042084:Paracoccus pantotrophus / Q000235:genetics / Q000378:metabolism; D033903:Periplasmic Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D003580:Cytochromes; D004269:DNA, Bacterial; D033903:Periplasmic Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006418:Heme; D010088:Oxidoreductases; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); C048228:nitrite reductase (heme)