CYCLIC VOLTAMMETRY AND H-1-NMR OF RHODOPSEUDOMONAS-PALUSTRIS CYTOCHROME C(2) - PH-DEPENDENT CONFORMATIONAL STATES

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c(2) from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E(o) values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted H-1-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pK(a) values of 2.5, 5.5, 6.6 and 9.0. The lowest pK(a) most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pK(a) is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pK(a) of 6.6 modulates the E(o) values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s(-1) at pH 9.2.