The binding of cyanide to cytochrome d in intact cells, spheroplasts, membrane fragments and solubilized enzyme from Salmonella typhimurium

Abstract

This investigation focused on the kinetics of cyanide binding to oxidized and reduced cytochrome d in Salmonella typhimurium intact cells, spheroplasts, membrane fragments and solubilized enzyme, and on the effect of pH on this binding. Cyanide bound to the oxidized form of cytochrome d under all experimental conditions, inducing a trough at 649 nm in the oxidized-cyanide-minus-oxidized difference absorption spectra. V max of cyanide binding to oxidized cytochrome d at pH 7.0 was 14.0±2.0 pmol/min/mg protein (prot.) in intact cells, 37.0±3.5 pmol/min/mg prot. in spheroplasts, 125.0±6.0 pmol/min/mg prot. in membrane fragments, and 538.0±8.5 pmol/min/mg prot. in solubilized cytochrome d. The pseudo-first order rate constants were 0.004 s −1 for intact cells, 0.005 s −1 for spheroplasts, 0.007 s −1 for membrane fragments and 0.025 s −1 for the solubilized enzyme. The V max value was highest at pH 7.0 for intact cells and solubilized cytochrome d and at pH 8.0 for both spheroplasts and membrane fragments. The K s of binding at pH 7.0 was around 4 mM in intact cells, spheroplasts and membrane fragments, but was 10.5 mM in solubilized cytochrome d. This difference between the K s values suggested a change in conformation, upon solubilization, leading to a decrease in the affinity of cyanide for the solubilized enzyme. The K s value was nearly the same at all pH investigated (pH 5–10). Cyanide was found to also bind to the reduced form of cytochrome d in membrane fragments ( K s=18±3 mM, V max=377±28 pmol/min/mg prot. at pH 7) and the solubilized enzyme ( K s=18±1.2 mM, V max=649±45 pmol/min/mg prot. at pH 7) with a lower affinity of cyanide for the reduced cytochrome d than for the oxidized enzyme. Pseudo-first order rate constants were 0.025 s −1 and 0.042 s −1 respectively for membrane fragments and solubilized enzyme. The value of V max for cyanide binding to the reduced cytochrome d, whether membrane-bound or solubilized, increased slightly with pH (for pH 6–10) while the K s value dropped significantly with increasing pH. The pH dependence observed here might be interpretable as a possible role for conformational transition associated with energy transduction. Finally, this investigation pointed to the influence of the microenvironment of a protein within the cell on its reactivity.