Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase

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Abstract
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., and 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 photoreduction 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase. Only catalytic amounts of deazaflavin are required, and the absorbance of this species in the region of the tryptophan emission is small. When the oxidase is reduced with deazaflavin, the uncorrected emission is at 328 nm, and the corrected spectrum is at 324 nm. We conclude that the 18-nm red shift of the tryptophan emission spectrum that is reported to occur when cytochrome oxidase is reduced, and ascribed specifically to the reduction of CuA [Copeland, R. A., Smith, P. A. and Chan, S. I. (1987) Biochemistry 26, 7311-7316; Copeland, R. A., Smith, P. A., and Chan, S. I. (1988) Biochemistry 27, 3552-3555], is due to the strong absorbance of dithionite in the region of the tryptophan emission spectrum. Our findings are presented as evidence against redox-linked, large-scale conformational changes within the cytochrome oxidase complex as possible intermediate steps in energy transduction.