Kinetics of the reduction of wild-type and mutant cytochrome c-550 by methylamine 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 conditions, the rate constants of the reactions have been determined as a function of the ionic strength, both for wild type cytochrome c-550 and for mutants in which the conserved residue Lys14 has been replaced as follows: Lys14-->Gln (mutant [K14Q]cytochrome c-550) and Lys14-->Glu (mutant [K14E]cytochrome c-550). The second-order rate constant of the reduction of cytochrome c-550 by MADH shows a biphasic ionic-strength dependence. At low ionic strength the rate constant remains unchanged (wild type) or increases ([K14Q]cytochrome c-550) with increasing ionic strength, while at high salt concentrations the rate constant decreases 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 slower than wild-type cytochrome c-550 by MADH, due to a lower association constant. It is concluded that MADH possesses a negative patch with which cytochrome c-550 associates. Lys14 plays an important role in the formation of the reaction complex. The midpoint potentials of wild-type and mutant cytochrome c-550 have been determined by using cyclic voltammetry. [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 show an increase in E0 of only 2 mV and 8 mV, respectively, compared to wild-type cytochrome c-550 (241 mV at pH 8.1). [K14Q]cytochrome c-550 and [K14E]cytochrome c-550 cytochrome c-550 are reduced by amicyanin at rates that are only slightly faster than for wild-type cytochrome c-550. The difference is partly attributable to the change in E0. High ionic strength results in a threefold increase in the rate in all three cases. These results indicate that charge interactions do not play a major role in the formation of the amicyanin/cytochrome c-550 reaction complex, suggesting an interaction at the hydrophobic patch of amicyanin. The reduction of cytochrome c-550 by MADH can be inhibited by Zn(2+)-substituted amicyanin. Ag(+)-amicyanin, however, has little effect on the reduction rate. These results suggest that MADH has a much higher affinity for Cu(2+)-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.