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 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.
Highlights
Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex
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 g1,2,3 ؍ 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 gmax signal at g ؍3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular
This work has provided the first purification and spectropotentiometric characterization of the NrfB cytochrome from any source. The genes for these novel pentaheme c-type cytochromes are only found in gene clusters that encode the NrfA pentaheme nitrite reductase in enteric members of the ␥-proteobacteria
Summary
Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex. The crystal structures of cytochrome c nitrite reductase from the sulfur-reducing bacterium Sulfurospirillum deleyianum, the closely related rumen bacterium Wolinella succinogenes, and the enteric bacterium E. coli have been determined recently [4, 7,8,9]. In the second group, which includes E. coli, nrfA clusters with genes encoding a putative periplasmic pentaheme cytochrome (nrfB), a periplasmic (4 ϫ [4Fe4S]) ferredoxin (nrfC) and an integral membrane putative quinol dehydrogenase (nrfD) [3, 5]. Despite the importance of periplasmic nitrite reduction to ammonium in enteric bacteria and the developing structure-informed biochemical understanding of the enzyme that catalyzes this process, the nature of electron delivery has never been addressed biochemically. In this paper we present the first purification and spectropotentiometric characterization of NrfB and demonstrate its competence as an electron donor to NrfA, with which it must transiently form a 20-heme [NrfB]2[NrfA]2 electron transfer complex
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