Redesign of the interior hydrophilic region of mitochondrial cytochrome c by site-directed mutagenesis.
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.
School of Chemical Sciences, University of East Anglia, Norwich, U.K.
A M
AM
Davies
J G
JG
Guillemette
M
M
Smith
C
C
Greenwood
A G
AG
Thurgood
A G
AG
Mauk
G R
GR
Moore
D001483:Base Sequence; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry; D004926:Escherichia coli; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D003574:Cytochrome c Group; D011994:Recombinant Proteins; C081433:heme propionate-7; D006418:Heme