The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 Å. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by (1) the fluorescence emission maximum at 328 nm and (2) the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the hemes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA. In contrast, the fluorescence spectral maximum of indole was shifted to a shorter wavelength near the critical micelle concentration of lauryl maltoside, indicating that indole had partitioned into the detergent micelle. The fluorescence spectrum of indole in a lauryl maltoside micelle resembles the tryptophan fluorescence of cytochrome c oxidase. These data support the idea that in a membrane protein there are two possible environments that may give rise to the fluorescence properties of tryptophan seen with cytochrome c oxidase: the interior of the protein and the protein/lipid or protein/detergent interface. The ability of Cs+ and I- to quench the fluorescence of indole in lauryl maltoside micelles is much greater than the ability of these agents as quenchers of the oxidase fluorescence. These quenching data suggest that in cytochrome c oxidase the fluorescent tryptophan residues are buried in the protein. Thus, quenching studies represent a method for distinguishing between the two possible types of hydrophobic environments in integral membrane proteins. It is concluded that the fluorescent tryptophans in cytochrome c oxidase are asymmetrically located in the complex, removed from the cytochrome c binding region, and buried in the interior of the protein rather than at the protein/lipid interface.
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