Purification and Spectropotentiometric Characterization of Escherichia coli NrfB, a Decaheme Homodimer That Transfers Electrons to the Decaheme Periplasmic Nitrite Reductase Complex

Abstract

Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically. This study reports the first purification of NrfB, a novel 20-kDa pentaheme c-type cytochrome encoded by the nrfB gene that follows the nrfA gene in many bacterial nrf operons. Analyses by gel filtration demonstrated that NrfB purifies as a decaheme homodimer. Analysis of NrfB by UV-visible and magnetic circular dichroism spectroscopy demonstrates that all five NrfB ferric heme irons are low spin and are most likely coordinated by two axial histidine ligands. Spectropotentiometry revealed that the midpoint redox potentials of five ferric hemes were in the low potential range of 0 to ؊400 mV. Analysis by low temperature EPR spectroscopy revealed signals that arise from two classes of bis-His ligated low spin hemes, namely a rhombic trio at g 1,2,3 ‫؍‬ 2.99, 2.27, and 1.5 that arises from two hemes in which the planes of histidine imidazole rings are near-parallel and a large g max signal at g ‫؍‬ 3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular. NrfB was also overexpressed as a recombinant protein, which had similar spectropotentiometric properties as the

X-ray structure of the membrane-bound cytochrome c quinol dehydrogenase NrfH reveals novel haem coordination

“…In NrfA proteins that interact with NrfB, there is also a conserved lysine. However, this residue should not be involved in NrfB haem ligation, as all the haems of this cytochrome are known to be bis‐histidinyl coordinated (Clarke et al , 2004). Moreover, NrfA and NrfB proteins form a transient complex.…”

Characterization of Shewanella oneidensis MtrC: a cell-surface decaheme cytochrome involved in respiratory electron transport to extracellular electron acceptors

“…At present, despite extensive efforts, the structure of MtrC is not known; however, primary structure analysis suggests that it, and also periplasmic decaheme MtrA [10], encodes two approximately 150 amino acid pentaheme modules with similarity to the pentaheme NrfB, an approximately 150 amino acid pentaheme periplasmic electron transport cytochrome widely involved in nitrite respiratory systems. All five hemes within NrfB are bishistidine-coordinated and it exhibits thermodynamic activity over a similar potential domain to MtrC [42].…”

Partial Functional Replacement of CymA by SirCD in Shewanella oneidensis MR-1

“…As has also been proposed for the iron sulfur proteins encoded by the sirC and mccC genes from S. oneidensis MR-1 and W. succinogenes, respectively, E. coli NrfC couples instead to a c-type cytochrome protein, NrfB (17,21,43). In E. coli, NrfD, via the iron sulfur protein NrfC, has been proposed to reduce the pentaheme c-type cytochrome NrfB via a putative NrfAB (20-heme) heterotetrameric complex in vivo (5,6). Interestingly, Clarke et al showed that a truncation of an N-terminal MtrA polypeptide, which shares significant homology and similar heme organization to NrfB from E. coli, forms a mature c-type cytochrome, supporting the idea that larger cytochromes may be modular extensions of smaller ones (7).…”