Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex.
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 native protein. Reconstitution experiments demonstrated that the reduced decaheme NrfB dimer could serve as a direct electron donor to the oxidized decaheme NrfA dimer, thus forming a transient 20-heme [NrfB](2)[NrfA](2) electron transfer complex.
Centre for Metalloprotein Spectroscopy and Biology, Schools of Biological Sciences and Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Thomas A
TA
Clarke
Victoria
V
Dennison
Harriet E
HE
Seward
Bénédicte
B
Burlat
Jeffrey A
JA
Cole
Andrew M
AM
Hemmings
David J
DJ
Richardson
D003574:Cytochrome c Group / Q000737:chemistry*; D045304:Cytochromes c / Q000502:physiology*; D019281:Dimerization; D004579:Electron Transport; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D006418:Heme / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D009572:Nitrite Reductases / Q000737:chemistry*; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000737:chemistry*; D013057:Spectrum Analysis
D003574:Cytochrome c Group; D029968:Escherichia coli Proteins; C492974:NrfB protein, E coli; D033903:Periplasmic Proteins; D006418:Heme; D045304:Cytochromes c; C022302:cytochrome C-552; D007501:Iron; D009572:Nitrite Reductases