The pH-induced isomerization of horse heart cytochrome c has been studied by tH NMR. We find that the transition occurring in D20 with a pKa measured as 9.5+0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0+0.6 s -~ at 27°C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are zlH~ = 12.8 +0.8 keal.moP, z/S~ = -12.9 __+ 2.0 e.u. for the forward reaction and ,Jilt = 6.0_+0.3 kcal.mo1-1, AS~= -35.1 + 1.3 e.u. for the reverse reaction (pH* =9.28).ztH ° and z/S ° for the isomerization are 6.7 + 0.6 kcal.mol -~ and 21.9 + 1.0 e.u., respectively.

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The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.

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