SEMIEMPIRICAL CALCULATIONS OF THE ELECTRONIC ABSORPTION-SPECTRA OF VITAMIN-B6 DERIVATIVES IN THE ACTIVE-SITE OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE

The electronic absorption spectra of derivatives of the vitamin B6-related cofactor of mitochondrial aspartate aminotransferase (mAspAT) have been calculated using a semiempirical, all-valence-electron INDO/S method with configuration interaction. Several models of partial active site structures were studied to assess the effects of the protein environment on the spectra of the coenzyme. The models include either of two forms of the cofactor, i.e., the pyridoxal 5'-phosphate (PLP) internal aldimine or the noncovalently bound pyridoxamine 5'-phosphate (PMP), and groups which model residues interacting directly or indirectly with PLP or PMP. Residues which are hydrogen bonded to the ring moiety of the cofactor include Asn 1 94, Asp222, and Tyr225. In addition, the indole of Trp140 could be involved in pi-stacking interactions with the pyridine ring. His143, Ser139, and a water molecule modulate the effect of Asp222 by forming an H-bonded network with this residue. The effects of these residues on the spectra of PLP and PMP were analyzed by calculations on models consisting of the cofactor and various combinations of the above residues. Geometries were taken from X-ray structural data, and hydrogen atom positions were optimized using the AM1 Hamiltonian. Results for both the protonated and unprotonated PLP aldimines are in good agreement with measured solution spectra. The calculations predict that the effect of the active site residues included in the model generally lead to a blue shift of the spectrum of the isolated cofactor. A further important result of these calculations is that, for a proper description of the effect of the charged Asp222 on the chromophore's spectrum, residues interacting with the former must also be included in the calculation. This appears to be primarily due to the delocalization of the charge due to the H-bonded network which links the charged species to the cofactor and other residues. The spectra for the PMP form show a similar response to the inclusion of active site residues as those for the PLP aldimine, although the quantitative agreement with experiment is not as satisfactory. The orientations of the calculated transition dipole moment vectors of the chromophore agree to within experimental error with the observed values. Effects of different protonation states and cofactor conformations on the spectra are also discussed.