Redox-related conformational changes in Rhodobacter capsulatus cytochrome c(2)

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the h eme proton assignments for Rhodobacter capsulatus ferricytochrome c(2). The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the H-1 and N-15 NMR spectra were obtained by a combination of H-1-H-1 and H-1-N-15 two-dimensi onal NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but w ith redox-related conformational changes between the two redox states. To u nderstand the decrease in stability of the oxidized state of this cytochrom e c(2) compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, p seudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the pos ition of some internal water molecules. Thus, it appears that the general d estabilization of cytochrorne c(2), which occurs on oxidation, is consisten t with the alteration of hydrogen bonds that result in changes in the inter nal dynamics of the protein.