QUENCHING OF INTRINSIC FLUORESCENCE OF YEAST CYTOCHROME-C PEROXIDASE BY COVALENTLY-BOUND AND NONCOVALENTLY-BOUND QUENCHERS

The intrinsic steady-state fluorescence of the heme enzyme cytochrome c peroxidase (CCP) has been characterized as a probe of its structure in solution. The fluorescence is dominated by tryptophan emission, which has a quantum yield of 7% relative to the tryptophan standard N-acetyltryptophanamide, and an emission maximum at 324 nm indicative of a relatively hydrophobic environment for the fluorescent residues. These fluorescence properties are consistent with the known structure of CCP; six of the seven tryptophan residues are well within quenching distance for efficient Forster energy transfer to the heme, so that the intrinsic fluorescence arises largely from Trp101 which is approximately 26 angstrom from the heme and partially buried. Quenching studies using Cs+, I-, and acrylamide are also consistent with this picture, since the charged species are poor quenchers, but acrylamide, which can penetrate the protein matrix, is a more effective quencher. The intrinsic fluorescence of two CCP derivatives with the quencher pentaammineruthenium(III) covalently attached to His6 and His60 has also been characterized. The His60 derivative, shown by X-ray analysis to be essentially structurally identical to native CCP, is 17% less fluorescent than native CCP, consistent with the quenching expected from distance calculations and the assignment of Trp101 as the major fluorescent center. The observed quenching of 38% in the second derivative is close to that predicted for ruthenation of His6 assuming that Trp101 is the major fluorophore. The fluorescence of compound 1 of CCP is also reported. This species, which has a ferryl (Fe(IV)=O) heme and a protein radical purportedly on Trp191, exhibits 9% higher fluorescence than native CCP. This increase can be accounted for by the 12-nm red-shift in the Soret band in compound I, which reduces the efficiency of Forster energy transfer. A similar fluorescence increase is seen for the cyanide adduct of ferric CCP which also has a red-shifted Soret relative to native CCP. Thus, there is no evidence from the intrinsic fluorescence of a global protein conformational change in CCP upon compound I formation, cyanide ligation, or ruthenation, in agreement with X-ray studies.