Fluorescence Quenching and Energy Transfer in Complexes between Horse‐Liver Alcohol Dehydrogenase and Coenzymes

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Abstract
The fluorescence properties of horse‐liver alcohol dehydrogenase were investigated with the aim of separating the contribution of Trp‐15 (which is close to the protein surface) from that of Trp‐314 (buried in the interior of the protein). Quenching of the protein fluorescence by iodide involves, to a larger degree, the longer wavelength region of the protein emission spectrum and is interpreted to involve only Trp‐15 (λmax at 340 nm and a quantum yield of 0.19). It is shown that quenching by NAD and NADH involves both types of tryptophan, but the ‘blue’ one to a larger extent. In the case of NADH, radiationless energy transfer between enzyme and reduced nicotinamide ring accounts for less than one half of the total protein fluorescence quenching. Energy transfer between the tryptophan and adenine rings is also possible, but it cannot account for the rest of the protein quenching. Thus, it is suggested that protein conformational changes, following NADH binding, are the cause of part of the fluorescence quenching. The extent to which quenching by NAD can be ascribed to radiationless energy transfer processes is also calculated. It is shown that despite the small spectral overlapping between coenzyme absorption and protein emission, the energy transfer contribution cannot be neglected. However, it is very likely that also in this case a sizeable part of the protein fluorescence quenching comes from protein conformational changes following coenzyme binding. The possible nature of these conformational changes is discussed, taking into account recent X‐ray data of enzyme‐coenzyme complexes.