title: Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2 pH-dependent conformational states.
abstract: The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E degree values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 1H-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 pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pKa most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E degree 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.
authors.affiliation: Department of Chemistry, University of Modena, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Battistuzzi
authors.forename: M
authors.initials: M
authors.lastname: Borsari
authors.forename: S
authors.initials: S
authors.lastname: Ferretti
authors.forename: M
authors.initials: M
authors.lastname: Sola
authors.forename: E
authors.initials: E
authors.lastname: Soliani
mesh_terms: D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D045362:Cytochromes c2; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012241:Rhodopseudomonas / Q000201:enzymology*
chemical_list: D003574:Cytochrome c Group; D045362:Cytochromes c2