Modulation of redox potential in electron transfer proteins: Effects of complex formation on the active site microenvironment of cytochrome b5
Abstract
The
reduction potential of cytochrome b5 is modulated ia 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 (∽40 mV shift) that is comparable,
if not larger, than the one originating from neutralization
of the charge on the solvent exposed heme propionate (∽30 mV shift).