The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

Abstract

The spectroscopic and functional properties of the single Met80Ala and double Tyr67His/Met80Ala mutants of human cytochrome c have been investigated in their ferric and ferrous forms, and in the presence of different ligands, in order to clarify the reciprocal effect of these two residues in regulating the access of exogenous molecules into the heme pocket. In the ferric state, both mutants display an aquo high spin and a low spin species. The latter corresponds to an OH -ligand in Met80Ala but to a His in the double mutant. The existence of these two species is also reflected in the functional behavior of the mutants. The observation that (i) a significant peroxidase activity is present in the Met80Ala mutants, (ii) the substitution of the Tyr67 by His leads to only a slight increase of the peroxidase activity in the Tyr67His/Met80Ala double mutant with respect to wild type, while the Tyr67His mutant behaves as wild type, as previously reported, suggests that the peroxidase activity of cytochrome c is linked to an overall conformational change of the heme pocket and not only to the disappearance of the Fe-Met80 bond. Therefore, in human cytochrome c there is an interplay between the two residues at positions 67 and 80 that affects the conformation of the distal side of the heme pocket, and thus the sixth coordination of the heme.

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

“…The most dramatic changes are observed in the fingerprint region, where specific iron‐ligand stretching vibrations corresponding to His18–Fe–His and His18–Fe–Lys misligation as well as a specific band pattern for His18–Fe–OH − and His18–Fe–H 2 O conformations are observed (see below). Therefore, this spectral region will be described in detail for each model compound, because it provides a useful marker for the identification of the non‐native conformations …”

“…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 . The effects of mutation on the structure and the peroxidase activity, which is induced by the CL interaction with the subsequent release of Cyt c into the cytosol where it acts as an apoptosis initiator, have also been reported . In this section, we will discuss and compare the effects of mutation of the conserved key residues Met80 and Tyr67 in Cyt c from different species: human, yeast, and horse heart.…”

“…The RR and UV–vis spectra of the ferric M80A mutant (Figure ), where the conserved Met80 ligand of the heme iron has been replaced by the non‐ligated Ala residue, of human, S. cerevisiae (yeast), and horse heart Cyt c are almost identical. At neutral pH, the UV–vis electronic absorption spectrum is characterized by the absence of the CT band at 695 nm and the Soret maximum at 408 nm.…”

“…Panels b and c: The intensities of the resonance Raman spectra are normalized to that of the ν 4 band (not shown). Reprinted from Ciaccio et al copyright (2017), with permission from Elsevier…”

Conference report—A report from the 5th International Taiwan Symposium on Raman Spectroscopy (TISRS 2017) and Taiwan Association of Raman Spectroscopy (TARS) Summer Camp

“…Dr. Giulietta Smulevich (Department of Chemistry “Ugo Schiff,” University of Florence, Italy) illustrated the structure–function relationship of human cytochrome c with the Tyr67 and Met80 ligand via UV–Vis absorption and resonance Raman spectroscopies . Dr. Shinsuke Shigeto (Department of Chemistry, Kwansei Gakuin University, Japan) studied the intermolecular vibration of biomolecules by low frequency Raman spectra and multivariate curve resolution using an alternate least‐squares fitting method.…”