Isolation and characterization of cytochrome c2 from Rhodopseudomonas palustris

Abstract

Cytochrome c 2 from Rhodopseudomonas palustris has been isolated and characterized by UV-Vis, electron paramagnetic resonance and NMR spectroscopy. The latter, through natural abundance 1H- 13C heteronuclear multiple quantum coherence experiments and homonuclear 2D spectra, allowed us to assign the signals of the heme substituents and of the iron-bound histidine and methionine. An analysis of the 13C chemical shift of the heme substituents has allowed us to localize the axes of the magnetic susceptibility tensor. The latter are consistent with the orientation of the two axial ligands s found from our NOE datae. he pH dependence of the shifts showed a p K a = 6.9 and the presence of a further p K a above 8.0. The present investigation shows how to appropriate use of NMR spectroscopy can provide a wealth of structural and electronic information even in the absence of any biochemical data.