ISOLATION AND CHARACTERIZATION OF CYTOCHROME C(2) FROM RHODOPSEUDOMONAS-PALUSTRIS

Cytochrome c(2) from Rhodopseudomonas palustris has been isolated and characterized by UV-Vis, electron paramagnetic resonance and NMR spect roscopy. The latter, through natural abundance H-1-C-13 heteronuclear multiple quantum coherence experiments and homonuclear 2D spectra, all owed us to assign the signals of the heme substituents and of the iron -bound histidine and methionine. An analysis of the C-13 chemical shif t of the heme substituents has allowed us to localize the axes of the magnetic susceptibility tensor. The latter are consistent with the ori entation of the two axial ligands as found from our NOE data. The pH d ependence of the shifts showed a pK(a) = 6.9 and the presence of a fur ther pK(a) above 8.0. The present investigation shows how the appropri ate use of NMR spectroscopy can provide a wealth of structural and ele ctronic information even in the absence of any biochemical data. (C) 1 998 Elsevier Science S.A.