Influence of Conserved Amino Acids on the Structure and Environment of the Heme of Cytochrome c₂. A Resonance Raman Study

Abstract

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.

Structural Characterization of Nitrosomonas europaea Cytochrome c‐552 Variants with Marked Differences in Electronic Structure

“…Although the X-ray crystal structure of R. capsulatus cyt c 2 reveals a nearly planar heme, [44] resonance Raman studies of this protein detect two components, one with significant heme ruffling, and the other with a more planar heme. [45] The basis for the sensitivity of this phenomenon to pH may be the influence of pH on hydrogen bonding within the Cys-X-X-Cys-His heme attachment motif, which modulates heme ruffling; this pH effect on heme conformation has been observed for heme c peptide fragments (microperoxidases). [40]…”

Structural and EPR Characterization of the Soluble Form of Cytochrome c-550 and of the psbV2 Gene Product from the Cyanobacterium Thermosynechococcus elongatus

“…Considering previous RR data on various ferric cytochromes and heme model compounds, the high-and lowfrequency regions of RR spectra of the psbV2 gene product are characteristic of a six-coordinated low-spin c-type heme (Desbois 1994, Döpner et al 1998, Hu et al 1993, Kitagawa et al 1975, Lefevre-Groboillot et al 2001, Othman and Desbois 1998, Othman et al 1994, Othman et al 1997, Verma et al 1988. However, the observed frequencies for the n 2 , n 3 , n 4 , n 8 , n 10 and n 50 modes are systematically higher when compared to those previously published for neutral ferric cyt c, cyt c 2 (His/ Met coordination) or cyt c 3 (His/His coordination) (Hu et al 1993, Kitagawa et al 1975, Othman et al 1997, Verma et al 1988. Among the available vibrational data, only the RR frequencies of an alkaline form of cyt c (His/hydroxide coordination) closely resemble those of the psbV2 gene product (Döpner et al 1998).…”

Nonplanar porphyrins and their significance in proteins

“…Thus, heme distortion provides a mechanism for differentiating CO and O 2 binding in hemoglobins and myoglobin. 31…”