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Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (1l is) as the postphotodissociation Cu'-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CUB to Fe,3 when CO binds to CUB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The timeresolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CUB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fe,3 pair. We speculate as to the identity ofthe ligand that is transferred between CUB and Fe,13 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.In a recent time-resolved infrared (TRIR) study (1) of the events after photodissociation of CO from cytochrome (cyt) a3 of reduced beef heart cytochrome oxidase (CcO), we reported conclusive evidence that photodissociated CO binds quantitatively to CuB at room temperature prior to equilibrating with solution. In a parallel kinetics study (6.E., P. M.

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime ca 7 microseconds) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.

The human lens is continually under photooxidative stress from ambient radiation. In the young lens the major absorbing (between 300-400 nm) species is the glucoside of 3-hydroxy kynurenine. Using time resolved fluorescence spectroscopy on both the isolated compound and the intact human lens, the first excited singlet state of this compound is shown to have fast (ps) decay processes. This would tend to minimize damage to lens constituents because there would be little time for energy transfer into more harmful channels. Thus, this compound appears to act as a protection for the retina. With aging, human lens proteins become yellow with absorption out to 450 nm. Time resolved diffuse reflectance spectroscopic studies on intact older human lenses showed that excitation (355 nm) resulted in the formation of long lived (microseconds) transient species with an absorption maximum at ca 490 nm. Similar spectra were obtained from two model systems used to explain age related changes in human lens proteins.