Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase.
We have measured the steady-state tryptophan fluorescence spectrum of cytochrome oxidase in its oxidized and fully reduced states. Reduction of the oxidized enzyme by sodium dithionite causes an apparent shift in the fluorescence emission maximum from 328 nm, in the oxidized enzyme, to 348 nm, in the reduced enzyme. This spectroscopic change has been observed previously and assigned to a redox-linked, conformational change in cytochrome oxidase [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. When dithionite-reduced enzyme sits in an open cuvette, the enzyme returns to the oxidized state, and the fluorescence maximum shifts back to 328 nm. However, the time course of the fluorescence change does not follow the redox state of the enzyme, monitored spectrophotometrically at 445,605, and 820 nm, but follows the disappearance of dithionite, which absorbs at 315 nm. Moreover, when the fluorescence emission spectrum of the dithionite-reduced enzyme is corrected for the absorbance due to dithionite, the fluorescence maximum is found 2 nm blue shifted, relative to that of the oxidized enzyme, at 326 nm. This dithionite-induced, red-shifted steady-state tryptophan fluorescence is also seen with the non-heme-containing enzyme carboxypeptidase A. The tryptophan emission spectrum of untreated carboxypeptidase A is at 332 nm, whereas in the presence of dithionite the emission spectrum of carboxypeptidase A is at 350 nm. When corrected for the absorbance of dithionite, the tryptophan emission maximum is at 332 nm. We have also used the photoreductant 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)
Ferreira-Rajabi
L
L
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Hill
B C
BC
D000818:Animals; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D002417:Cattle; D004227:Dithionite / Q000032:analysis; D003576:Electron Transport Complex IV / Q000032:analysis*; D066298:In Vitro Techniques; D008929:Mitochondria, Heart / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000032:analysis*
D004227:Dithionite; D014364:Tryptophan; D003576:Electron Transport Complex IV; D002268:Carboxypeptidases; D043422:Carboxypeptidases A