Modulation of redox potential in electron transfer proteins: Effects of complex formation on the active site microenvironment of cytochrome b(5)

The reduction potential of cytochrome b(5) is modulated via the formation o f a complex with polylysine at the electrode surface (Rivera et al., Bioche mistry, 1998, 37, 1485). This modulation is thought to originate from the n eutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutrali zation of the charge on the heme propionate contributes to the modulation o f the redox potential of cytochrome b(5) has been obtained, evidence demons trating that water exclusion from the complex interface plays a similar rol e has not been conclusive. Herein we report the preparation of the V45I/V61 I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b (5). This mutant has been engineered with the aim of restricting water acce ssibility to the exposed heme edge of cytochrome b(5). The X-ray crystal st ructure of the V45I/V61I mutant revealed that the side chain of Ile at posi tions 45 and 61 restricts water accessibility to the interior of the heme c avity and protects a large section of the heme edge from the aqueous enviro nment. Electrochemical studies performed with the V45I/V61I mutant of cytoc hrome b(5), and with a derivative in which the heme propionates have been c onverted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the redu ction potential of cytochrome b(5). In fact, these studies showed that excl usion of water from the complex interface exerts an effect (similar to 40 m V shift) that is comparable, if not larger, than the one originating from n eutralization of the charge on the solvent exposed heme propionate (similar to 30 mV shift).