respectively, are of comparable energy (eq 4 and ref 36). In fact, the "doubly excited" state was found50 to be the lowest excited singlet state in some systems with a small HOMO-LUMO energy gap «, and a large number of photoreactions initiated in the St (50) B.

Two high resolution crystal structures of cytosolic aspartate aminotransferase from pig heart provide additional insights into the stereochemical mechanism for ligand-induced conformational changes in this enzyme. Structures of the homodimeric native structure and its complex with the substrate analog 2-methylaspartate have been refined, respectively, with 1.74-Å x-ray diffraction data to an R value of 0.170, and with 1.6-Å data to an R value of 0.173. In the presence of 2-methylaspartate, one of the subunits (subunit 1) shows a ligandinduced conformational change that involves a large movement of the small domain (residues 12-49 and 327-412) to produce a "closed" conformation. No such transition is observed in the other subunit (subunit 2), because crystal lattice contacts lock it in an "open" conformation like that adopted by subunit 1 in the absence of substrate. By comparing the open and closed forms of cAspAT, we propose a stereochemical mechanism for the open-to-closed transition that involves the electrostatic neutralization of two active site arginine residues by the negative charges of the incoming substrate, a large change in the backbone (,) conformational angles of two key glycine residues, and the entropy-driven burial of a stretch of hydrophobic residues on the N-terminal helix. The calculated free energy for the burial of this "hydrophobic plug" appears to be sufficient to serve as the driving force for domain closure.

To establish the state of protonation of quinonoid species formed nonenzymically from pyridoxal phosphate (PLP) and diethyl aminomalonate, we have studied absorption spectra of the rapidly established steady-state mixture of species. We have evaluated the formation constant and the spectrum of the mixture of Schiff base and quinonoid species. For N-methyl-PLP a singly protonated species with a peak at 464 nm is formed from the unprotonated aldehyde and the conjugate acid of diethyl aminomalonate with a formation constant Kf of 240 M-1. The very intense absorption band with characteristic vibrational structure (most evident as a shoulder at 435 nm) is accompanied by a weaker, structured band at about 380 nm and a weak, broad band at 330 nm. We suggest that the 380-nm band may represent a tautomeric form of the quinonoid compound. Protonation of the phosphate group appears to affect the spectrum only slightly. The corresponding mixture of Schiff base and quinonoid species formed from PLP has a very similar spectrum at pH 6-7. It has a formation constant Kf of 230 M-1 and a pKa of 7.8, which must be attributed to the ring nitrogen atom. The dissociated species, which may be largely carbanionic, has a strong structured absorption band at 430 nm and a weaker one, again possibly a tautomer, in the 330-nm region. The analysis establishes that in all species a proton remains on either the phenolic oxygen or the imine nitrogen. Proton NMR spectroscopy, under some conditions, reveals only two components: free PLP and what appears to be Schiff base. However, we suggest that the latter may, in fact, be a quinonoid form, either alone or in rapid equilibrium with the Schiff base. Absorption spectra of quinonoid species formed in enzymes are analyzed and compared with the spectra of the nonenzymic species.

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We have fitted over 160 electronic absorption spectra of over 95 aromatic compounds and some other substances with lognormal distribution curves to evaluate the shapes of the bands. The compounds include simple benzene derivatives, phenols, anilines, benzaldehyde, benzoic acid, nitrobenzene, and potassium permanganate. The well-defined n-π* transitions of acetone, benzaldehyde, nitrobenzene, and pyrazine are included. Effects of solvents have been studied in a number of cases. The fitting of absorption bands with lognormal curves is compared with resolution with Gaussian curves or with other types of skewed Gaussian curves. We conclude that the lognormal curve provides an excellent approximation to the shapes of spectral bands in aromatic as well as many other organic and inorganic compounds. The ability to analyze complex spectra by resolution with lognormal curves may be usefully applied in a number of ways.