The reduction potential of
cytochrome b5 is modulated via the formation of a complex with
polylysine at the
electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a
solvent exposed
heme propionate and from
dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the
heme propionate contributes to the modulation of the redox potential of
cytochrome b5 has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM)
cytochrome b5. This mutant has been engineered with the aim of restricting water accessibility to the exposed
heme edge of
cytochrome b5. The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the
heme cavity and protects a large section of the
heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of
cytochrome b5, and with a derivative in which the
heme propionates have been converted into the corresponding dimethyl
ester groups, clearly demonstrate that
dehydration of the
heme edge contributes to the modulation of the reduction potential of
cytochrome b5. In fact, these studies showed that exclusion of water from the complex interface exerts an effect (approximately 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the
solvent exposed
heme propionate (approximately 30 mV shift).