Electric Field and Conformational Effects of Cytochrome c and Solvent on Cytochrome c Peroxidase Studied by High-Resolution Fluorescence Spectroscopy

Abstract

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.

Study on the interaction between salicylic acid and catalase by spectroscopic methods

“…Fluorescence spectrometry is the often used and effective method to study the interaction of protein with exogenous substances [16]. Similar to other proteins, the Tyr, Trp and Phe residues of CAT result in its endogenesis fluorescence.…”

Protonation of porphyrin in iron-free cytochromec: Spectral properties of monocation free base porphyrin, a charge analogue of ferric heme

“…Protein charge effects have been reported for spectra of chlorophyllide in myoglobin 29 and porphyrin in cytochrome c peroxidase. 30 The redox of cyt c is reported to be independent of pH from the range of 4 to 8 31 but it would be interesting to examine in more detail whether there are also subtle pH effects on the redox in this range.…”

Effect of a Protein Electric Field on the CO Stretch Frequency. Finite Difference Poisson−Boltzmann Calculations on Carbonmonoxycytochromesc

“…52 Recently, the FDPB method was used to simulate the electrostatic field imposed by cytochrome c peroxidase on porphyrin cytochrome c. Ionic strength effects were included, and a qualitative correlation was established with the fluorescence properties of the iron-depleted cytochrome. 3 Another study, investigating vibrational dynamics in the photosynthetic reaction center, used the FDPB method to calculate the electric field at each of the reaction center's carbonyls and estimated vibrational spectral shifts on the order of -2 to 2 cm -1 , which were invoked to explain observed intensity increases in the amide transitions. 38,39 In this work, we used the FDPB method to correlate experimentally observed CO vibrational frequencies with electrostatic potential and electric field calculations performed on the CO ligand and on protein residues.…”