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 yea st iso-1-cytochrome c destabilizes the native conformation of the protein b y facilitating the ligand exchange reactions that are associated with the a lkaline conformational transitions of the ferricytochrome. Of the Phe82 var iants surveyed thus far, Phe82Trp is unique in that it adopts a thermodynam ically stable, high-spin conformation at mildly alkaline pH. This species e xhibits spectroscopic features that can only be detected transiently in oth er ferricytochromes c within the first 100 ms immediately after a pH-jump f rom neutrality to pH >10. Spectroscopic characterization of this high-spin reaction intermediate suggests that in addition to an obligatory pentacoord inate heme iron, a group within the heme pocket coordinates the heme iron b ut 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.