Influence of conserved amino acids on the structure and environment of the heme of cytochrome c2. A resonance Raman study.
Resonance Raman spectra using Soret excitations of oxidized and reduced Rhodobacter capsulatus cytochrome c2 at pH 7.5 were studied. The spectra of oxidized cytochrome c2 show three components for the v10 mode at 1638, 1633, and 1629 cm(-1). The intensities of these components are sensitive to the excitation wavelength. This effect is explained in the context of a conformational equilibrium of the ferriheme between a nearly planar structure and two ruffled structures. In the case of reduced cytochrome c2, the absolute frequencies as well as the excitation-dependent frequency dispersion of the v10 mode (1618-1621 cm(-1)) indicate a displacement of the conformational equilibrium of heme toward the more planar structures. To measure the influence of some key amino acid residues on the heme-protein interaction of cytochrome c2, four site-directed mutants of Rb. capsulatus cytochrome c2 have been studied by resonance Raman spectroscopy and their spectra compared with the spectra obtained for the wild type cytochrome. The mutants studied are K14E/K32E, P35A, W67Y, and Y75F. The spectral changes induced by the mutations are interpreted in terms of alterations in the structure and/or environment of the cytochrome c2 heme in the framework of the expected role of the different amino acid residues in the stability and redox potential.
Othman
S
S
Département de Biologie Cellulaire et Moléculaire, CEA et CNRS-URA 2096, CEA/Saclay, Gif-sur-Yvette, France.
Fitch
J
J
Cusanovich
M A
MA
Desbois
A
A
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics / Q000502:physiology*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045362:Cytochromes c2; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
D000596:Amino Acids; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D006418:Heme; D045362:Cytochromes c2