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

Shilpa Bali, Martin J. Warren, Stuart J. Ferguson

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

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 languageEnglish
Pages (from-to)4944-4955
Number of pages12
JournalFEBS Journal
Volume277
Issue number23
DOIs
Publication statusPublished - Dec 2010

Keywords

  • Cytochrome cd
  • d heme biosynthesis
  • Denitrification
  • Nitrite reductase
  • Paracoccus pantotrophus
  • Tetrapyrrole

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