ELECTRIC-FIELD AND CONFORMATIONAL EFFECTS OF CYTOCHROME-C AND SOLVENTON CYTOCHROME-C PEROXIDASE STUDIED BY HIGH-RESOLUTION FLUORESCENCE SPECTROSCOPY

Electronic spectra of mesoporphyrin-substituted yeast cytochrome c per oxidase (MP-CcP) were measured as a function of pH, ionic strength, an d binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the v ibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widt hs 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 10 3, and 16 203 cm-1. MP-CcP at pH 8 could be analyzed using two compone nts with peak positions at 16 048 and 16 193 cm-1. The disappearance o f 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 dista l side of the porphyrin. Computer simulations of the electrostatic fie ld that CcP imposes on its porphyrin show that, in the presence of cha rged axial histidines His52 and His175, the electrostatic field at por phyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent s pectral shifts of the 0,0 positions of the resolved components, althou gh hydrogen bonding may also affect these positions. On the other hand , the peak position of the components was not affected by ionic streng th or binding of cyt c, implying that the electrostatic field of the h eme pocket of MP-CcP remains unchanged. Indeed, computed changes in io nic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength a nd 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 vibration al frequencies due to the nonsymmetry of the porphyrin and/or that tau tomers experience various distortions. Comparison of the vibrational s pectrum 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.