Spectroscopic and electronic structure studies of metalloenzyme active sites.

摘要

This work reports on the electronic and geometric structural studies of xanthine oxidoreductase (XO), S-ribosylhomocysteinase (LuxS) and inducible nitric oxide synthase (iNOS) active sites resulting from the joint applications of electronic absorption, electron paramagnetic resonance and magnetic circular dichroism spectroscopic methods.;XO catalyzes formal oxygen atom insertion into a substrate C-H bond, but differs from monooxygenase enzymes in that the inserted oxygen atom derives from metal activated water and reducing equivalents are generated rather than consumed. Studies on aldehyde "inhibited" XO (a paramagnetic form observed during XO catalysis), analyzed in terms of the relationships between the g-, 95,97Mo hyperfine, and the 13C hyperfine tensors have provided structural insights into the nature of substrate/product bound at the Mo active site. The results indicate that aldehyde "inhibited" is a tetrahedral analogue of the calculated transition state in XO catalytic mechanism.;S-ribocylhomocysteinase (LuxS) catalyzes the non-redox cleavage of a stable thioether bond, a difficult reaction from chemist's perspective. This metalloenzyme plays a key role in quorum sensing which makes its investigation an attractive target for inhibition and development of novel antibacterial agents. This study utilized Co(II)-d7 substituted tetrahedral LuxS. Thus, analysis for g-, 59Co hyperfine and zfs (D and E) tensors of wild-type, mutants (C84A and C84D) and relevant small molecule analogues, (PATH)CoBr and (PATH)CoNCS have provided a detailed description of LuxS active site. The results indicate that the LuxS active site is a distorted tetrahedral with approximate C3V geometry and the catalytic reaction begins by the substrate displacing water.;The iNOS catalyzes the oxidation of L-arginine to a signaling molecule, NO and L-citrulline with NADPH and O2 as cosubstrates. The emerging evidence suggests that the production of NO is facilitated by the interdomain electron transfer from the FMN to the catalytic heme site. This work reports a comparative spectroscopic study of wild-type and mutant proteins of a human iNOS bidomain oxygenase/FMN construct. The results indicate notable effects of mutations in the adjacent FMN domain on the heme structure suggesting that the conserved surface residues in the FMN domain (E546 and E603) play key roles in facilitating a productive alignment of the FMN and heme domains in iNOS.