Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex.
The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.
Centre for Molecular and Structural Biochemistry, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Colin
C
Lockwood
Julea N
JN
Butt
Thomas A
TA
Clarke
David J
DJ
Richardson
D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009572:Nitrite Reductases / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D016415:Sequence Alignment
D003574:Cytochrome c Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; C492974:NrfB protein, E coli; D006418:Heme; D045304:Cytochromes c; C022302:cytochrome C-552; D009572:Nitrite Reductases