Proton electron-nuclear double-resonance spectra of molybdenum(V) in different reduced forms of xanthine oxidase.
Electron-nuclear double-resonance (ENDOR) spectra of protons coupled to molybdenum(V) in reduced xanthine oxidase samples have been recorded. Under appropriate conditions these protons may be studied without interference from protons coupled to reduced iron-sulfur centers. Spectra have been obtained for the molybdenum(V) species known as Rapid, Slow, Inhibited, and Desulfo Inhibited. Resonances corresponding to at least nine protons or sets of protons are observed for all four species, with coupling constants in the range 0.08-4 MHz. Most of these protons do not exchange when 2H2O is used as solvent. Additional protons giving couplings up to 40 MHz are also detected. These correspond to EPR-detectable protons studied in earlier work. The strongly coupled protons may be replaced by 2H, through appropriate use of 2H2O or of 2H-substituted substrates, with consequent disappearance of the 1H resonances. In most cases the corresponding 2H ENDOR features have also been observed. The nature of the various coupled protons is briefly discussed. Results permit specific conclusions to be drawn about the structures of the Inhibited and Desulfo Inhibited species. In particular, the data indicate that the aldehyde residue of the Inhibited species has been oxidized and that the four protons derived from the ethylene glycol molecule in the Desulfo Inhibited species are not all equivalent. Recent assignments [Edmondson, D.E., & D'Ardenne, S.C. (1989) Biochemistry 28, 5924-5930] of the weakly coupled protons in the latter species appear not to be soundly based. The possibility of obtaining more detailed structural information from the spectra is briefly considered.(ABSTRACT TRUNCATED AT 250 WORDS)
Howes
B D
BD
School of Chemistry and Molecular Sciences, University of Sussex, Brighton, U.K.
Pinhal
N M
NM
Turner
N A
NA
Bray
R C
RC
Anger
G
G
Ehrenberg
A
A
Raynor
J B
JB
Lowe
D J
DJ
D004578:Electron Spin Resonance Spectroscopy; D008982:Molybdenum; D010084:Oxidation-Reduction; D011522:Protons; D014969:Xanthine Oxidase
D011522:Protons; D008982:Molybdenum; D014969:Xanthine Oxidase