Structural studies of yeast Iso‐1 cytochrome c mutants by resonance Raman spectroscopy

Abstract

The Ser82 and Phe82 variants of yeast iso-I cytochrome c were studied by resonance Raman spectroscopy. In both oxidation states, distinct spectral changes were observed for some of those bands in the low-frequency region, which sensitively respond to conformational perturbations of the protein environment of the heme. These bands can be assigned to modes which include strong contributions of vibrations largely localized in the propionatecarrying pyrrole rings A and D. This indicates structural differences in the deeper part of the heme crevice, remote from the mutation site. This conclusion is in line with previous results from X-ray crystallography and NMR spectroscopy. No differences in the resonance-Raman spectra were observed which can be directly correlated with conformational changes of the heme pocket in the vicinity of the mutation site. Temperature-dependent resonance Raman experiments of the oxidized mutants revealed spectral changes which are closely related to those observed for cytochrome c upon adsorption to charged silver surfaces by surface-enhanced resonance Raman spectroscopy. These spectral changes can be attributed to an opening of the heme crevice accompanied by a weakening of the iron-methionine ligand bond. The temperature-dependent conformational transition occurs at approximately 30°C for the Ser82 variant and at about 45°C for the Phe82 variant, implying that the Phe + Ser substitution significantly lowers the thermal stability of the heme pocket. The reduced forms of both mutants are stable up to 65 "C.

Structural changes in cytochrome c upon hydrogen-deuterium exchange

“…The present data show that in contrast to the ~442/450-cm™1 doublet the bands at 371 and 379 cm™1 are not affected upon H/D exchange (except for a slight narrowing of the 371-cm™1 band). These findings as well as the arguments given in our previous work (Hildebrandt, 1990(Hildebrandt, ,1991Hildebrandt et al, 1991Hildebrandt et al, ,1992 led us to retain the original assignment of the two bands between 440 and 450 cm™1. However, a definite assignment will only be possible based on RR data of Cyt with isotopically labeled the spectrum is dominated by a broad and asymmetric peak at -685 cm-1 which exhibits several shoulders and smaller bands on both wings of the envelope.…”

“…. The assignment of these bands has been discussed elsewhere(Hildebrandt et al, 1991). At this point, we focus on the bands at 442.1 and 448.8 cm™1 which we have attributed to porphyrin modes, including major contributions from the propionate bending vibrations.…”

“…The disappearance of these bands has been shown to be a sensitive marker for a conformational state of cytochrome c in which the heme pocket assumes an open structure (Hildebrandt, 1991). On the basis of a variety of indirect evidence, this band has been assigned to a porphyrin mode including a major contribution from the bending vibrations of the propionate side chains of the heme (Hildebrandt, 1991;Hildebrandt et al, 1991Hildebrandt et al, , 1992.…”

Probing the non‐native states of Cytochrome c with resonance Raman spectroscopy: A tool for investigating the structure–function relationship

“…The power of site‐directed mutagenesis combined with structural probes to illuminate the molecular interactions at the heme active site has been well known since the marked sensitivity of the RR spectrum to alterations in the surrounding side chains of CCP was reported . The study of many variants of Cyt c has been devoted to (a) the identification of the predominant non‐native ligand in the five different states of the unfolded Cyt c, (b) to understand the structural and functional role of conserved residues in the interaction between Cyt c and Cyt c oxidase, and (c) the structure–stability relationship of the protein . Recently, following the discovery of the role of Cyt c in apoptosis, many studies have been devoted to understanding the mechanism of the CL interaction by studying the effect of mutation of key residues on the interaction .…”

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

“…Considering the N-acetylmethionine complex of MP8 as a model compound of the His/Met coordination of heme c, its ν 50 frequency is decreased from 358 to 354 cm -1 when the heme iron is reduced (Othman, 1994). For yeast iso-1cyt c, the heme reduction decreases the ν 50 frequency from 361 to 356 cm -1 (Hildebrandt et al, 1991). Therefore, the normal trend of the ν 50 mode is a decrease in its frequency by 4-5 cm -1 upon heme reduction.…”