The role of the internal hydrogen bond network in first‐order protein electron transfer between Saccharomyces cerevisiae iso‐1‐cytochrome c and bovine microsomal cytochrome b5

Abstract

An internal water molecule (designated WAT166) is found in iso‐1‐cytochrome c which is part of a redox‐state‐dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can be altered or even displaced by site‐directed mutagenesis leading to structural perturbations and associated changes in redox potential. Using saturation transfer 1H‐NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso‐1‐cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild‐type or variant yeast iso‐1‐cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome b5. For three variants of yeast iso‐1‐cytochrome c the rate constants measured by saturation transfer are wild‐type (Asn52, Eo= 270 mV, kex= 0.3 s‐1), Asn52 → Ala (Eo= 240 mV, kex= 0.6 s‐1), Asn52 →Ile(Eo=220 mV, kex=1.0 s‐1). The first‐order rates are compared with that of a fourth variant Phe82 → Gly which has been measured previously (Eo=220 mV, kex=0.7 s‐1). An analysis of the variation in the observed cross exchange rate using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both G* and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins. Copyright © 1991, Wiley Blackwell. All rights reserved