The polarity of tyrosine 67 in yeast iso-1-cytochrome c monitored by second 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 derivative spectroscopy. Yeast iso-1-cytochrome c contains five tyrosines, one 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 differentiate events that were occurring in or near the heme pocket from those occurring closer to the protein's surface. The wild-type protein shows a substantial change in the second derivative spectrum as the protein goes from oxidized to reduced; mutants lacking tyrosine 67 do not show this change. This indicates that it is primarily the spectrum of tyrosine 67 that changes as the protein cycles between the oxidized and reduced state. One thing that contributes to the overall polarity of the heme pocket is a water molecule hydrogen bonded to several of the nearby residues. The wild-type protein has one water molecule in the heme pocket but this can be increased or decreased by introducing mutations into the protein. N52A has two water molecules and N52I has no water molecule in the pocket. The three proteins allowed us to assess the contribution of water to the inferred heme crevice polarity. The number of water molecules in the crevice correlates with the perceived polarity 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 and hydrogen bonding pattern of the amide it replaces.
Schroeder
H R
HR
Department of Biology, Concordia University, Montreal, QC, Canada.
McOdimba
F A
FA
Guillemette
J G
JG
Kornblatt
J A
JA
D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D045304:Cytochromes c; D006736:Horses; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D045304:Cytochromes c