Structure and Spectroscopy of the Periplasmic Cytochrome c Nitrite Reductase from Escherichia coli

Abstract

The crystal structure and spectroscopic properties of the periplasmic penta-heme cytochrome c nitrite reductase (NrfA) of Escherichia coli are presented. The structure is the first for a member of the NrfA subgroup that utilize a soluble penta-heme cytochrome, NrfB, as a redox partner. Comparison to the structures of Wolinella succinogenes NrfA and Sulfospirillum deleyianum NrfA, which accept electrons from a membrane-anchored tetra-heme cytochrome (NrfH), reveals notable differences in the protein surface around heme 2, which may be the docking site for the redox partner. The structure shows that four of the NrfA hemes (hemes 2-5) have bis-histidine axial heme-Fe ligation. The catalytic heme-Fe (heme 1) has a lysine distal ligand and an oxygen atom proximal ligand. Analysis of NrfA in solution by magnetic circular dichroism (MCD) suggested that the oxygen ligand arose from water. Electron paramagnetic resonance (EPR) spectra were collected from electrochemically poised NrfA samples. Broad perpendicular mode signals at g similar 10.8 and 3.5, characteristic of weakly spin-coupled S = 5/2, S = 1/2 paramagnets, titrated with E(m) = -107 mV. A possible origin for these are the active site Lys-OH(2) coordinated heme (heme 1) and a nearby bis-His coordinated heme (heme 3). A rhombic heme Fe(III) EPR signal at g(z) = 2.91, g(y) = 2.3, g(x) = 1.5 titrated with E(m) = -37 mV and is likely to arise from bis-His coordinated heme (heme 2) in which the interplanar angle of the imidazole rings is 21.2. The final two bis-His coordinated hemes (hemes 4 and 5) have imidazole interplanar angles of 64.4 and 71.8. Either, or both, of these hemes could give rise to a "Large g max" EPR signal at g(z)() = 3.17 that titrated at potentials between -250 and -400 mV. Previous spectroscopic studies on NrfA from a number of bacterial species are considered in the light of the structure-based spectro-potentiometric analysis presented for the E. coli NrfA.

The relationship between redox enzyme activity and electrochemical potential—cellular and mechanistic implications from protein film electrochemistry

“…Thus, for NrfA it appears that the attenuation of activity at low potentials reflects formal reduction of a site positioned some distance from the active site. 52 One mechanism by which a change of oxidation state could change the rate of a reaction is through a change in the products of reduction. Multiple products have been reported for reduction of nitrite by various heme proteins.…”

Structural Basis of Redox-coupled Protein Substrate Selection by the Cytochrome c Biosynthesis Protein ResA

“…Many bacteria contain multi-heme c-type cytochromes in which the hemes have electron transfer and catalytic activities, e.g. cytochrome c nitrite reductase (2,3). Many bacteria also contain enzymes with heme c and at least one other redox cofactor, e.g.…”

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

“…The fifth heme is attached to the novel CXXCK motif that is essential for catalysis (6). 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 all three structures NrfA crystallized as a homodimer, with the hemes within each monomer closely packed to form arrangements of near-parallel and near-perpendicular heme pairs.…”

“…In all three structures NrfA crystallized as a homodimer, with the hemes within each monomer closely packed to form arrangements of near-parallel and near-perpendicular heme pairs. In the absence of substrate, the NrfA active site heme displays a distal lysine ligand and proximal water or hydroxide ligand (8,9).…”

“…Clearly, electron transfer from quinol to the NrfA in the different groups is distinct. The different protein-protein and cofactor-cofactor interactions implicit in this situation may be reflected by insertions and deletions in loop regions of the polypeptide chain in the two subgroups that can be identified in primary and tertiary structure analyses (9). 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.…”