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The reaction mechanism of the molybdoenzyme xanthine oxidase has been further investigated by 13C and 17O ENDOR of molybdenum(V) species and 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 side reaction with aldehyde substrates, and (iii) Alloxanthine, a species formed by reaction of reduced enzyme with the inhibitor, alloxanthine. The Very Rapid signal was developed either with [8-13C]xanthine or with 2-oxo-6-methylpurine using enzyme equilibrated with [17O]H2O. The Inhibited signal was developed with 2H13C2HO and the Alloxanthine signal by using [17O]H2O. Estimates of Mo-C distances were made, from the anisotropic components of the 13C-couplings, by corrected dipolar coupling calculations and by back-calculation from assumed possible structures. Estimated distances in the Inhibited and Very Rapid species were about 1.9 and less than 2.4 A, respectively. A Mo-C bond in the Inhibited species is very strongly suggested, presumably associated with side-on bonding to molybdenum of the carbonyl of the aldehyde substrate. For the Very Rapid species, a Mo-C bond is highly likely. Coupling from a strongly coupled 17O, 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 Alloxanthine species. That the coupled oxygen of the Very Rapid species is the one that appears in the product uric acid molecule was confirmed by new kinetic data. It is concluded that this oxygen of the Very Rapid species does not, as frequently assumed, originate from the oxo group of the oxidized enzyme. A new turnover mechanism is proposed, not involving direct participation of the oxo ligand group, and based on that of Coucouvanis et al. [Coucouvanis, D., Toupadakis, A., Lane, J. D., Koo, S. M., Kim, C. G., Hadjikyriacou, A. (1991) J. Am. Chem. Soc. 113, 5271-5282]. 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 vicinity of molybdenum and perhaps a ligand of it) on the bound substrate carbon, to give an intermediate that on intramolecular one-electron oxidation gives the Very Rapid species. The latter, in keeping with the 13C, 17O, and 33S couplings, is presumed to have the 8-CO group of the uric acid product molecule bonded side-on to molybdenum, with the sulfido molybdenum ligand retained, as in the oxidized enzyme.

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Burns & Bulen (1965) showed that cell-free extracts of Azotobacter vinelandii contained an ATP-activated system capable of fixing nitrogen. When provided with sodium dithionite and an ATPgenerating system, their preparations evolved less hydrogen under nitrogen than under argon, the difference being accountable as nitrogen reduced to ammonia. Hardy & Knight (1966b) showed that dithionitedependent adenosine-triphosphatase activity was associated with nitrogen fixation. More recently preparations from A. vinetandii and Cloetridium pa8teurianum have been found to reduce a variety of additional substrate including acetylene (Dilworth, 1966), nitrous oxide, N3-and CN-ions (Hardy & Knight,1966a). We now report that nitrogen-fixing extracts of Azotobacter chroococcum reduce not only CN-ions but also methyl isocyanide, and indicate the relevance of these reactions to the mechanism of nitrogen fixation. Methods and Materials. Active cell-free extracts from A. chroococcum (N.C.I.B. 8003) grown on the nitrogen-free sucrose medium of Burk, Newton, Wilson & Harris (1953) were obtained by disruption at pH7-4 with ultrasound by using a Dawe Soniprobe. Whole cells and debris were removed by centrifuging at 18000g for 30min. and the supernatant was heated for 7min. at 600 under an atmo

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