THE POLARITY OF TYROSINE-67 IN YEAST ISO-1-CYTOCHROME-C MONITORED BY 2ND DERIVATIVE SPECTROSCOPY

The average tyrosine polarity of 10 yeast iso-1-cytochrome c proteins and two horse heart cytochrome c proteins was assayed by second deriva tive spectroscopy. Yeast iso-1-cytochrome c contains five tyrosines, o ne of which (tyrosine 67) is in the heme pocket. The wild-type protein and the Y67F, N52V Y67F, and N521 Y67F proteins were used to differen tiate events that were occurring in or near the heme pocket from those occurring closer to the protein's surface. The wild-type protein show s a substantial change in the second derivative spectrum as the protei n goes from oxidized to reduced; mutants lacking tyrosine 67 do not sh ow this change. This indicates that it is primarily the spectrum of ty rosine 67 that changes as the protein cycles between the oxidized and reduced state. One thing that contributes to the overall polarity of t he heme pocket is a water molecule hydrogen bonded to several of the n earby residues. The wild-type protein has one water molecule in the he me pocket but this can be increased or decreased by introducing mutati ons into the protein. N52A has two water molecules and N52I has no wat er molecule in the pocket. The three proteins allowed us to assess the contribution of water to the inferred heme crevice polarity. The numb er of water molecules in the crevice correlates with the perceived pol arity of the pocket when one takes account of the fact that the second water molecule in the crevice of the N52A mutant takes the position a nd hydrogen bonding pattern of the amide it replaces.