Evidence for a Proximal Histidine Interaction in the Structure of Cytochromes c‘ in Solution: A Resonance Raman Study

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Abstract
Soret-excited resonance Raman (RR) spectra of oxidized and reduced cytochromes c‘ from Rhodospirillum molischianum and Rhodobacter sphaeroides, in solution, are reported. The spectra of the type I ferricytochromes c‘ in both species contain different extents of two forms. One of these is readily assignable to a “normal” five-coordinated high-spin heme. The second species with v3 and v10 modes at 1502 and 1635 cm-1, respectively, is attributed to a five-coordinated intermediate-spin heme. The RR data show that the equilibrium between these two forms is species-dependent at neutral pH and 20 °C. The v(Fe−His) mode of the a form of reduced cytochromes c‘ is assigned to a band at 228−231 cm-1, indicating that the proximal His has a strong electronegative character. X-ray crystallographic data on R. molischianum ferricyt c‘ show that the proximal His has no interaction with either the protein or water molecules [Finzel, B. C., Weber, P. C., Hardman, K. D., & Salemme, F. R. (1985) J. Mol. Biol. 186, 627−643]. Considering that the absence of H bonding at the coordinated histidine corresponds to a low frequency for the v(Fe−His) mode (195−205 cm-1), the structure and/or environment of the proximal histidine appears different for cyt c‘(III) in the crystal and cyt c‘(II) in aqueous solution. To account for the elevated frequency of the v(Fe−His) mode of cyt c‘(II), several possibilities have been examined. Among these, we propose that a conserved Lys residue, located in the protein sequence three residues before the His ligand, can form an electrostatic interaction with the (His)N1 atom, directly or through a water molecule. It is further suggested that this electrostatic interaction could also play a role in the high-spin ↔ intermediate-spin equilibrium of oxidized cytochromes c‘.