1H NMR investigation of cytochrome cd1: complexes with electron-donor proteins.
Mixtures of the dissimilatory nitrite reductase cytochrome cd1 from Pseudomonas aeruginosa and potential electron-donating proteins were prepared in both fully oxidized and fully reduced states and examined by 1H NMR spectroscopy. The relatively narrower lines of the donor proteins enabled them to be clearly observed in spectra in the presence of significant amounts of the high molecular weight cd1. Mixtures of the physiological donor (Pseudomonas ferrocytochrome c-551) and ferrocytochrome cd1 showed specific line-broadening effects on the resonances of c-551 that depended on the mole ratio of c-551 to cd1. The experimental broadening fit a model in which c-551 is in intermediate or fast exchange between free solution and a complex with cd1, with an association constant for the complex in excess of 10(4) M-1. The model yields a minimum estimate for the forward bimolecular rate constant of 5 X 10(7) M-1 s-1 and suggests that the actual value may be much larger. The complexation was independent of pH in the range of 6-8, was independent of ionic strength over a salt concentration range of 20-1000 mM, and possessed a low thermal activation barrier. Mixtures of ferricytochrome c-551 and ferricytochrome cd1 showed no observable NMR perturbations, indicating that any hypothetical complex involving the oxidized forms must follow different dynamical and/or equilibrium conditions. No observable NMR perturbations existed in spectra of mixtures of cd1 and mammalian cytochrome c or Pseudomonas azurin in either oxidation state.
D000818:Animals; D003574:Cytochrome c Group / Q000378:metabolism; D003580:Cytochromes; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006736:Horses; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008970:Molecular Weight; D009206:Myocardium; D009572:Nitrite Reductases; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D003574:Cytochrome c Group; D003580:Cytochromes; D006859:Hydrogen; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming); D003576:Electron Transport Complex IV