Electric field and conformational effects of cytochrome c and solvent on cytochrome c peroxidase studied by high-resolution fluorescence spectroscopy.
Electronic spectra of mesoporphyrin-substituted yeast cytochrome c peroxidase (MP-CcP) were measured as a function of pH, ionic strength, and binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the vibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widths of their 0,0 bands. The composite 0,0 band of MP-CcP at pH 6 could be resolved into three components with peak positions at 16,046, 16,103, and 16,203 cm-1. MP-CcP at pH 8 could be analyzed using two components with peak positions at 16,048 and 16,193 cm-1. The disappearance of the 16,103-cm-1 component at alkaline pH suggests that it is due to a "chemical substate" arising from protonation of His52 in the distal side of the porphyrin. Computer simulations of the electrostatic field that CcP imposes on its porphyrin show that, in the presence of charged axial histidines His52 and His175, the electrostatic field at porphyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent spectral shifts of the 0,0 positions of the resolved components, although hydrogen bonding may also affect these positions. On the other hand, the peak position of the components was not affected by ionic strength or binding of cyt c, implying that the electrostatic field of the heme pocket of MP-CcP remains unchanged. Indeed, computed changes in ionic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength and binding of cyt c to MP-CcP is on the relative contributions of the components, suggesting some rearrangements in the vicinity of the heme. Finally, shifts in the position of the vibrational lines for MP-CcP components indicate either that the tautomers have different vibrational frequencies due to the nonsymmetry of the porphyrin and/or that tautomers experience various distortions. Comparison of the vibrational spectrum of the first excited singlet state of mesoporphyrin in CcP and horseradish peroxidase also suggests that the heme pocket in the two peroxidases provides different steric restrictions.
Anni
H
H
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia 19104-6089.
Vanderkooi
J M
JM
Sharp
K A
KA
Yonetani
T
T
Hopkins
S C
SC
Herenyi
L
L
Fidy
J
J
D001665:Binding Sites; D003198:Computer Simulation; D003574:Cytochrome c Group / Q000378:metabolism / Q000494:pharmacology*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D009994:Osmolar Concentration; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature
D003574:Cytochrome c Group; D012997:Solvents; D006639:Histidine; D003578:Cytochrome-c Peroxidase