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Active site substituted Cd(II) horse liver alcohol dehydrogenase has been studied by Perturbed Angular Correlation of Gamma rays Spectroscopy during turnover conditions for benzaldehyde and 4-trans-(N,N-dimethylamino)cinnamaldehyde. The ternary complex between alcohol dehydrogenase NAD+ and Cl-, and the binary complex between alcohol dehydrogenase and orthophenanthroline have also been studied. The Nuclear Quadrupole Interaction parameters have been interpreted in terms of different coordination geometries for Cd(II) in the catalytic zinc site of the enzyme. Calculation of the nuclear quadrupole interaction for cadmium in the catalytic site of the enzyme with and without coenzyme, based upon the four coordinated geometries determined from X-ray diffraction, agrees with the experimentally determined values. The ternary complexes between enzyme, NAD+ and either Cl- or trifluoroethanol and the binary complex between enzyme and orthophenanthroline have almost identical spectral parameters which are not consistent with a four coordinated geometry, but are consistent with a five coordinated geometry. The non-protein ligands for the ternary complex with trifluoroethanol are suggested to be an alkoxide group and a water molecule. The Nuclear Quadrupole Interaction parameters for the productive ternary complex between enzyme, NADH and an aldehyde is consistent with the four coordinated geometry predicted from X-ray diffraction data having the carbonyl group of the aldehyde substituting the water molecule as ligand to the metal.

We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-photon excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single-and two-photon excitation were essentially identical. The steady-state limiting anisotropy r 0 of PPO in glycerol at −5 °C measured for twophoton excitation is significantly higher than that observed for one-photon excitation. The r 0 value of 0.54 for two-photon excitation is well in excess of the theoretical maximum of 0.4 for singlephoton excitation. A similar value of r 0 ≃ 0.50 was obtained from the frequency-domain anisotropy data with two-photon excitation of PPO in methanol, butanol, and propylene glycol at 20 °C. These higher values of r 0 indicate that two-photon excitation results in a more highly oriented photoselected population, which can increase the resolution of rotational correlation times and/or complex anisotropy decays. The anisotropy resolution can still be increased by using global analysis of anisotropy decays measured with single-and two-photon excitation.

The small electron transporting copper protein, azurin, has been studied in order to investigate the interplay between the oxidation states of the metal and its coordination geometry. The results show that the metal coordination geometry for Ag(I) in Ag(I) substituted wild type azurin is only slightly different from the geometry for Cd(II) in cadmium substituted azurin both being similar to the geometry for copper in native azurin. Furthermore, the coordination geometry for Ag(I) in the Met121 to Leu substituted mutant of azurin is also similar to the geometry of copper in native azurin. In contrast, previously published results show that Cd(II) substituted Met121Leu-azurin exhibits two different coordination geometries for Cd(II), one again similar to the wild type geometry and another very flexible and distinctly different from wild type azurin. These results have been obtained by Perturbed Angular Correlation of γ-rays spectroscopy using the two radioactive isotopes 111Ag(I) and 111mCd(II) as probes of a monovalent and a divalent ion, respectively. The technique also revealed that the metal-coordination geometry for Ag(I) in wild type azurin relaxes to the coordination geometry of Cd(II) on a time scale of 100 ns after the decay from 111Ag(I) to 111Cd(II). We suggest that the role of Met121 is to maintain the rigid tricoordinated metal coordination geometry independent of the oxidation state of the metal.