CYTOCHROME C(553), FROM DESULFOVIBRIO-VULGARIS (HILDENBOROUGH) - ELECTROCHEMICAL PROPERTIES AND ELECTRON-TRANSFER WITH HYDROGENASE

An electrochemical study of the periplasmic cytochrome c(553) of Desul fovibrio vulgaris (Hilden-borough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic volt ammetry. The voltammograms obtained were reversible and revealed that this cytochrome showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25 degrees C was found to be 62 mV versus the normal hydrogen electrode. It was observed tha t the temperature dependence was discontinuous with a transition tempe rature at 32 degrees C. The standard reaction entropy at the growth te mperature of the organism (37 degrees C) was calculated to be Delta S degrees' = -234 J mol(-1) K-1. The pH dependence of the midpoint poten tial could be described with one pK of the oxidized form with a value of 10.6. The second-order rate constant for electron transfer between cytochrome c(553) and the Fe-hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c(3) and hydrogenase was measured for comparison. The secon d-order rate constants are 2X10(7)M(-1) s(-1) for cytochrome c(553) an d 2X10(8)M(-1) s(-1) for cytochrome c(3). The kinetic parameters of th e hydrogenase for both cytochromes were determined using the spectroph otometric hydrogen consumption assay. With cytochrome c(553) this resu lted in a K-m of 46 mu M and a maximum turnover number of 7.1x10(2) s( -1) in the H-2 consumption assay. The values with cytochrome c(3) were 17 mu M and 6.4X10(2) s(-1), respectively. The importance of the diff erent kinetic parameters for contrasting models proposed to describe t he function of the Fe-hydrogenase are discussed.