Characterization of an alkaline transition intermediate stabilized in the Phe82Trp variant of yeast iso-1-cytochrome c.
In general, mutation of the phylogenetically conserved residue Phe82 in yeast iso-1-cytochrome c destabilizes the native conformation of the protein by facilitating the ligand exchange reactions that are associated with the alkaline conformational transitions of the ferricytochrome. Of the Phe82 variants surveyed thus far, Phe82Trp is unique in that it adopts a thermodynamically stable, high-spin conformation at mildly alkaline pH. This species exhibits spectroscopic features that can only be detected transiently in other ferricytochromes c within the first 100 ms immediately after a pH-jump from neutrality to pH >10. Spectroscopic characterization of this high-spin reaction intermediate suggests that in addition to an obligatory pentacoordinate heme iron, a group within the heme pocket coordinates the heme iron but is then replaced either by Met80, to revert to the native conformation, or by Lys73 or Lys79, to yield one of the conventional alkaline conformers. Evidence is presented to suggest that this group is either a hydroxide ion or Tyr67 rather than a loosely bound Met80.
Rosell
F I
FI
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Harris
T R
TR
Hildebrand
D P
DP
Döpner
S
S
Hildebrandt
P
P
Mauk
A G
AG
D019943:Amino Acid Substitution; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D010649:Phenylalanine; D014364:Tryptophan; D045304:Cytochromes c; D007501:Iron