INFLUENCE OF CONSERVED AMINO-ACIDS ON THE STRUCTURE AND ENVIRONMENT OF THE HEME OF CYTOCHROME C(2) - A RESONANCE RAMAN-STUDY

Resonance Raman spectra using Soret excitations of oxidized and reduce d Rhodobacter capsulatus cytochrome c(2) at pH 7.5 were studied. The s pectra of oxidized cytochrome c(2) show three components for the v(10) mode at 1638, 1633, and 1629 cm(-1). The intensities of these compone nts are sensitive to the excitation wavelength. This effect is explain ed in the context of a conformational equilibrium of the ferriheme bet ween a nearly planar structure and two ruffled structures. In the case of reduced cytochrome c(2), the absolute frequencies as well as the e xcitation-dependent frequency dispersion of the v(10) mode (1618-1621 cm(-1)) indicate a displacement of the conformational equilibrium of h eme toward the more planar structures. To measure the influence of som e key amino acid residues on the heme-protein interaction of cytochrom e c(2), four site-directed mutants of Rb. capsulatus cytochrome ct hav e been studied by resonance Raman spectroscopy and their spectra compa red with the spectra obtained for the wild type cytochrome. The mutant s studied are K14E/K32E, P35A, W67Y, and Y75F. The spectral changes in duced by the mutations are interpreted in terms of alterations in the structure and/or environment of the cytochrome c(2) heme in the framew ork of the expected role of the different amino acid residues in the s tability and redox potential.