Role of arginine-38 in regulation of the cytochrome c oxidation-reduction equilibrium

Abstract

Arg-38 is an internal residue of mitochondrial cytochrome c that is close to heme propionate-7. Previous work comparing the behavior of cytochromes c from several species [Moore, G. R., Harris, D. E., Leitch, F. A., & Pettigrew, G. W. (1984) Biochim. Biophys. Acta 764, 331-342] has suggested that Arg-38 lowers the pKa of this propionate group and thereby accounts for the relative pH independence of the cytochrome c reduction potential from pH 5 to pH 8. The influence of Arg-38 on the oxidation-reduction equilibrium of yeast iso-1-cytochrome c has now been investigated by electrochemical, NMR, and theoretical analysis of six specifically mutated forms of this protein in which Arg has been replaced by Lys, His, Gln, Asn, Leu, or Ala. As the electron-withdrawing character of the residue at position 38 decreases, the reduction potential of the protein also decreases, with the largest decrease (ca. 50 mV) observed for the Ala variant. However, the variation in the reduction potentials of the mutants as a function of pH was similar to that observed for the wild-type protein. The effects of some of these mutations on the pKa values of His-33 and His-39 have been determined by NMR spectroscopy and found to be minimal. Calculations of the electrostatic free energy for the Leu-38 variant predict a decrease in the reduction potential of this mutant that is remarkably close to that observed experimentally. This work establishes that while Arg-38 contributes to the relatively high reduction potential of cytochrome c, this residue does not appear to be the sole functionality responsible for lowering the heme propionate-7 pKa.

Modeling of protein conformational fluctuations in pKa predictions

“…Our calculation results suggest that the following nine groups (all with pK a values outside the range from 3 to 11 by more than one pK unit) may not titrate in this pH range: Arg13, 38 and 91, Lys55, 79 and 87, Tyr48, Asp90, and the C terminus. Cutler et al (1989) have used NMR spectroscopy to study the proton titration of yeast iso-1-ferricytochrome c. The assigned pK a for His39, 6.6, is close to that determined by Barker et al (1991), 6.5, and to our prediction, 6.8. The assigned pK a for His33, 6.8, is also reproduced by our calculations (predicted pK a 6.4).…”

Arginine residues at internal positions in a protein are always charged

“…Our studies do not contribute definite insight into mechanisms for stabilization of charged Arg in a nonaqueous environment, but they do show that the Arg side chain has a unique capacity to retain its charge in environments that should be expected to promote the neutral form. This ability of Arg to stay charged appears to be fundamental for its functional role as a voltage sensor (8)(9)(10), as the essential filter in aquoporins (18,30,31), and in other proteins that perform fundamental energy transduction processes (4,(11)(12)(13).…”

“…Arginine (Arg) is the ionizable amino acid that is found most frequently buried in the protein interior (1)(2)(3). Arg residues in relatively hydrophobic environments are essential in a variety of biological processes, such as regulation of conformation or redox potentials (4,5), viral capsid assembly (6), electrostatic steering (7), voltage sensing across lipid bilayers (8)(9)(10), H þ transport (4,(11)(12)(13), and peptide translocation across bilayers (14,15). Internal Arg residues also play critical roles at protein-protein interfaces (3,16), in enzyme active sites (1,3,17), and in a variety of transport channels (18,19).…”

Structure–Function Relationships in Heme-Proteins

“…Structure-function relationships on the control of redox properties have been investigated for over 30 years with the greatest efforts centered on cytochrome c (Moore et al, 1984;Pielak et al, 1985;Cutler et al, 1989;Brayer and Murphy, 1996;Rafferty et al, 1996). Nevertheless, and despite substantial structural data from numerous c-type cytochromes, including, for example, c 2 , c 5 , c 6 , c 550 , c 551 , c 552 , c 553 , the attempts to accurately predict the redox potentials have had limited success.…”