Proton‐NMR studies of the effects of ionic strength and pH on the hyperfine‐shifted resonances and phenylalanine‐82 environment of three species of mitochondrial ferricytochrome c

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3‐CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso‐1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the ß‐CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3‐CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870–7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3‐CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 β‐CH2 group and the heme 3‐CH3 substituent. In addition, studies of the effect of pH on the 1H‐NMR spectrum of yeast iso‐1 ferricytochrome c show that the heme 3‐CH3 proton resonance exhibits a pH‐dependent shift with an apparent pK in the range of 6.0 –7.0. The chemical shift change of the yeast iso‐1 ferricytochrome c heme 3‐CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934–4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C‐terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3‐CH3 group in yeast iso‐1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8‐CH3 and the 7α‐CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of ∼ 7.0. The titrating group responsible for these spectral changes is proposed to be His39.