Quenching of intrinsic fluorescence of yeast cytochrome c peroxidase by covalently- 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 Förster energy transfer to the heme, so that the intrinsic fluorescence arises largely from Trp101 which is approximately 26 A 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 I of CCP is also reported. This species, which has a ferryl (FeIV = O) heme and a protein radical purportedly on Trp191, exhibits 9% higher fluorescence than native CCP.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
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Fox
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Ferreira-Rajabi
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Hill
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English
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D002586:Cesium / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D005453:Fluorescence; D007454:Iodides / Q000737:chemistry; D012428:Ruthenium / Q000737:chemistry; D017975:Ruthenium Compounds; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry
D000178:Acrylamides; D003486:Cyanides; D007454:Iodides; D017975:Ruthenium Compounds; D002586:Cesium; D020106:Acrylamide; C063246:pentaammineruthenium(3+); D012428:Ruthenium; D014364:Tryptophan; D003578:Cytochrome-c Peroxidase