Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis

Abstract

Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent. Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it. To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe. For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured. All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins. The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein. Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe. The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions.

Design of metal cofactors activated by a protein–protein electron transfer system

“…This result indicates that the midpoint potentials of 1⅐HO, 2⅐HO, and 3⅐HO are influenced by the surrounding protein environment, rather than the substituent effect on the ligand (6). The redox potential of cytochrome c was reported to be influenced by charge contribution of the heme 7-propionate (28). Thus, we propose that the large negative redox potentials of Schiff-base complexes, 1 and 2, in HO could arise from the negative charge of carboxylate.…”

The Methanol-induced Globular and Expanded Denatured States of Cytochrome : A Study by CD Fluorescence, NMR and Small-angle X-ray Scattering

“…We note that the far-UV CD spectrum at 25% methanol is surprisingly similar to that of the anion-induced molten globule state, and that at 60% methanol there are stronger negative peaks at 208 nm and 222 nm and a positive peak at about 190 nm compared with the native state (0% methanol). We note that the near-UV CD spectra for 25% and 60% methanol do not have sharp negative peaks at 282 nm and 288 nm typical for the native state, which have been assigned to Trp59 (Davies et al, 1993), indicating that methanol destroys the tertiary structure.…”

Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis

“…Arg-38 affects the redox behaviors of Cc by charge-charge interactions with the heme posterior propionate and͞or by hydrogen bonding to the heme posterior propionate mediated by surrounding water molecules, namely W125 and W139 (22,27,33,34). The Arg38Ala mutant of yeast iso-1 Cc shows a significant increase of its reduction potential (Ϸ40 mV) and a more stable oxidized form compared to the wild-type yeast iso-1 Cc (34). Our experiments also demonstrate that T-Cc has a much lower rate of reduction by ascorbate than S-Cc and hh-Cc ( Table 2).…”