C = C stretch as well as a phenyl C = C stretch, but it is still thought that much of the remaining line width is inhomogeneous because of the presence of conformers. The excited-state spectrum is very similar to the ground-state spectrum of the irans-stilbene radical anion. This similarity is appealing because both molecules have an electron in an antibonding 7Γ-7Γ* orbital. This interpreta tion immediately explains the observed frequency shifts: The C = C olefinic bond is weakened, and the ethylenic carbon phenyl carbon single bond is strengthened. This pioneering study il lustrates the power of picosecond reso nance Raman spectroscopy in elucidat ing the structure and dynamics of elec tronically excited states. As our final example we present a study of the reaction of cytochrome ox idase with oxygen, in which a combina tion of rapid mixing and pulse-probe time-resolved resonance Raman spec troscopy is used to detect intermedi ates in a chemical reaction of biological interest. Cytochrome oxidase catalyzes the reduction of oxygen to water. It is a mechanistically complex four-elec tron/four-proton reaction involving oxygen binding at one Fe 2 + —Cu + site followed by electron transfer from this site and a distant Fe 2 + —Cu + site. Im portant questions include the nature of the initial oxygen adduct and partially reduced intermediates. To address these questions, Babcock and co-workers rapidly mixed a solu tion of cytochrome oxidase, in which the heme binding site was blocked with carbon monoxide, with oxygenated buffer (19). The 532-nm second har monic from a 7-ns pulsed Nd:YAG la ser was used to photodissociate the carbon monoxy-heme complex, making the binding site available for oxygen. At variable delay times, a 416-nm probe pulse, generated by Stokes-shifting in H 2 of the 355-nm third harmonic, was used to excite the resonance Ra man scattering, which was detected by a 0.3-m single spectrograph and an in tensified photodiode array. Figure 4 shows the time evolution of the spectrum. The 10-ns spectrum shows both an oxidation state marker (1355 cm - 1 ) and a formyl stretching frequency (1666 cm - 1 ) that prove that the reduced deoxy enzyme has been generated. The shift of the oxidation state marker to higher frequency and the decrease in intensity of the formyl stretching mode as the reaction pro ceeds indicate that the protein is being oxidized. The surprising feature of the data is that little change is observed over the first 50 μ& of the reaction, dur ing which optical experiments had clearly established that oxygen addi tion and partial oxidation of the metal centers had occurred. This mystery was
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