KINETICS OF THE REDUCTION OF WILD-TYPE AND MUTANT CYTOCHROME C-550 BYMETHYLAMINE DEHYDROGENASE AND AMICYANIN FROM THIOBACILLUS-VERSUTUS

To elucidate the kinetic properties of the methylamine dehydrogenase ( MADH) redox chain of Thiobacillus versutus the reduction of cytochrome c-550 by MADH and amicyanin has been studied. Under steady state cond itions, the rate constants of the reactions have been determined as a function of the ionic strength, both for wild type cytochrome c-550 an d for mutants in which the conserved residue Lys14 has been replaced a s follows: Lys14-->Gln (mutant [K14Q] cytochrome c-550) and Lys14-->Gl u (mutant [K14E]cytochrome c-550). The second-order rate constant of t he reduction of cytochrome c-550 by MADH shows a biphasic ionic-streng th dependence. At low ionic strength the rate constant remains unchang ed (wild type) or increases ([K14Q]cytochrome c-550) with increasing i onic strength, while at high salt concentrations the rate constant dec reases monotonically as the ionic strength increases. It is suggested that conformational freedom exists in the association complex and that this is favourable for electron transfer. [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 are reduced at rates 20-fold and 500-fold slowe r than wild-type cytochrome c-550 by MADH, due to a lower association constant. It is concluded that MADH possesses a negative patch with wh ich cytochrome c-550 associates. Lys14 plays an important role in the formation of the reaction complex. The midpoint potentials of wild-typ e and mutant cytochrome c-550 have been determined by using cyclic vol tammetry. [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 show an in crease in E(0) of only 2 mV and 8 mV, respectively, compared to wild-t ype cytochrome c-550 (241 mV at pH 8.1). [K14Q]cytochrome c-550 and [K 14E]cytochrome c-550 cytochrome c-550 are reduced by amicyanin at rate s that are only slightly faster than for wild-type cytochrome c-550. T he difference is partly attributable to the change in E(0). High ionic strength results in a threefold increase in the rate in all three cas es. These results indicate that charge interactions do not play a majo r role in the formation of the amicyanin/cytochrome c-550 reaction com plex, suggesting an interaction at the hydrophobic patch of amicyanin. The reduction of cytochrome c-550 by MADH can be inhibited by Zn2+-su bstituted amicyanin. Ag+-amicyanin, however, has little effect on the reduction rate. These results suggest that MADH has a much higher affi nity for Cu2+-amicyanin (substrate) than for Cu+-amicyanin (product). On the basis of these findings the roles of the components of the MADH redox chain are discussed.