Numbers and exchangeability with water of oxygen-17 atoms coupled to molybdenum (V) in different reduced forms of xanthine oxidase.

The effect of using [17O]water (24-50% enriched) as solvent on the Mo(V) electron paramagnetic resonance spectra of different reduced forms of xanthine oxidase has been investigated. All the Mo(V) signals are affected. Procedures are described, based on the use of difference spectral techniques, that facilitate interpretation of such spectra. The number of coupled oxygen atoms may be determined by estimation of the fraction of the spectrum that remains unchanged by the isotope at a known enrichment. For a species having two coupled oxygen atoms, the use of two different isotope enrichments permits elimination from the difference spectra of the contribution of the two singly substituted species. From the application of these methods, it is concluded that not only the strength of the hyperfine coupling of oxygen ligands of molybdenum but also their number and their exchangeability with the solvent vary from one reduced form of the enzyme to another. The inhibited species from active xanthine oxidase has been studied in the most detail. It has two weakly coupled oxygen atoms [A(17O)av = 0.1-0.2 mT] that do not exchange with the solvent. A cyclic structure is proposed for this species in which two oxygen ligands of molybdenum are bonded to the carbon of the formaldehyde or other alcohol or aldehyde molecule that reacted in producing the signal. Structures of the other signal-giving species from active xanthine oxidase (Very Rapid and Rapid types 1 and 2) are discussed, as is corresponding information on species from the desulfo enzyme and from sulfite oxidase.