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 l igation state of the heme iron in cytochrome c(2) from Rhodopseudomona s palustris were monitored by electrochemical and spectroscopic measur ements. In the pH range 1.5-11, the E(o) values (and/or the peak poten tials) determined by cyclic voltammetry, the electronic spectra and th e 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, whi ch is accompanied by a conformational change. The equilibrium with an apparent pK(a) of 6.6 modulates the E(o) values without determining an y detectable spectral change and most likely involves the acid/base eq uilibrium of an histidine residue in close vicinity of the heme (possi bly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (mos t probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic cons tant of 0.98 s(-1) at pH 9.2.