EVIDENCE FAVORING MOLYBDENUM-CARBON BOND FORMATION IN XANTHINE-OXIDASE ACTION - O-17 AND C-13-ENDOR AND KINETIC-STUDIES

The reaction mechanism of the molybdoenzyme xanthine oxidase has been further investigated by C-13 and O-17 ENDOR of molybdenum(V) species a nd by kinetic studies of exchange of oxygen isotopes. Three EPR signal -giving species were studied: (i) Very Rapid, a transient intermediate in substrate turnover, (ii) Inhibited, the product of an inhibitory s ide reaction with aldehyde substrates, and (iii) Alloxanthine, a speci es formed by reaction of reduced enzyme with the inhibitor, alloxanthi ne. The Very Rapid signal was developed either with [8-C-13]xanthine o r with 2-oxo-6-methylpurine using enzyme equilibrated with [O-17]H2O. The Inhibited signal was developed with (HCHO)-H-2-C-13-H-2 and the Al loxanthine signal by using [O-17]H2O. Estimates of Mo-C distances were made, from the anisotropic components of the C-13-couplings, by corre cted dipolar coupling calculations and by back-calculation from assume d possible structures. Estimated distances in the Inhibited and Very R apid species were about 1.9 and less than 2.4 Angstrom, respectively. A Mo-C bond in the Inhibited species is very strongly suggested, presu mably associated with side-on bonding to molybdenum of the carbonyl of the aIdehyde substrate. For the Very Rapid species, a Mo-C bond is hi ghly likely. Coupling from a strongly coupled O-17, not in the form of an oxo group, and no coupling from other oxygens was detected in the Very Rapid species. No coupled oxygens were detected in the Alloxanthi ne species. That the coupled oxygen of the Very Rapid species is the o ne that appears in the product uric acid molecule was confirmed by new kinetic data. It is concluded that this oxygen of the Very Rapid spec ies does not, as frequently assumed, originate from the oxo group of t he oxidized enzyme. A new turnover mechanism is proposed, not involvin g direct participation of the oxo ligand group, and based on that of C oucouvanis et al. [Coucouvanis, D., Toupadakis, A., Lane, J. D., Koo, S. M., Kim, C. G., Hadjikyriacou, A. (1991) J. Am. Chem. Soc. 113, 527 1-5282]. It involves formal addition of the elements of the substrate (e.g., xanthine) across the Mo=S double bond, to give a Mo(VI) species . This is followed by attack of a ''buried'' water molecule (in the vi cinity of molybdenum and perhaps a ligand of it) on the bound substrat e carbon, to give an intermediate that on intramolecular one-electron oxidation gives the Very Rapid species. The latter, in keeping with th e C-13, O-17, and S-33 couplings, is presumed to have the 8-CO group o f the uric acid product molecule bonded side-on to molybdenum, with th e sulfide molybdenum ligand retained, as in the oxidized enzyme.