Characterisation of the electron self-exchange rates in hexametaphosphate-cytochrome-c aggregates measured using high-resolution 1H-NMR spectroscopy.

Abstract

1H-NMR spectroscopy has been used to measure the rate of unimolecular electron exchange between cytochrome c molecules in protein aggregates stabilised by the addition of sodium hexametaphosphate. The average intracomplex electron exchange rate is measured from line broadening of hyperfine-shifted resonances of ferricytochrome c in an equimolar mixture of reduced and oxidised protein. The line-broadening due to electron exchange is significantly greater than that due to protein aggregation and reaches a maximum value between 1-2 mol hexametaphosphate/mol protein. Significantly the exchange-induced broadening is a first-order process and is directly proportional to the size of the cytochrome c oligomer. From the temperature dependence of exchange broadening the activation enthalpy was estimated to be 75.8 kJ mol-1 whereas the activation entropy was 295 J mol-1 K-1 for a dimer of cytochrome c at a hexametaphosphate/protein molar ratio of 1. Both activation parameters decrease in magnitude as the order of the cytochrome c oligomer increases. The rates of intracomplex electron exchange in Saccharomyces cerevisiae iso-2 and Candida krusei cytochromes c are lower than that of the horse protein, implying that primary sequence plays a fundamental part in determining the rate of exchange. The relevance of these observations is discussed in terms of the function of cytochrome c.