Preparation of Aldehyde Oxidase in Its Native and Deflavo Forms

The Mononuclear Molybdenum Enzymes

“…The UV/vis absorption spectrum and the spectral change observed on reduction of either the native or deflavo form of aldehyde oxidase from rabbit liver is essentially indistinguishable from that shown in Figure for milk xanthine oxidase. , As with xanthine dehydrogenase from liver sources, earlier preparations of aldehyde oxidase yielded small amounts (∼5% of the enzyme molybdenum) of a “resting” Mo(V) EPR signal that apparently arises from enzyme that has been inactivated by treatment with organic solvent. It is possible to obtain enzyme in a form that is devoid of this signal. ,, The “rapid type 2” Mo(V) EPR signal is readily seen with aldehyde oxidase and is quite similar to that seen with xanthine oxidase ( g 1,2,3 = 1.9895, 1.9701, 1.9624; a av = 14 and 8.3 G for the two strongly coupled protons). , The second proton is coupled less strongly in the “type 2” signal in aldehyde oxidase than is seen with xanthine oxidase, however, and the signal-giving species seen in aldehyde oxidase is considered to be intermediate in some conformational sense to the species giving the “type 1” and “type 2” signals in xanthine oxidase.…”

Properties of the prosthetic groups of rabbit liver aldehyde oxidase: a comparison of molybdenum hydroxylase enzymes

“…The concentrations of all enzyme species except "rapid" Mov were related to the concentration of total enzyme active centers. The proportion of enzyme centers which contained functional Mo was determined on the basis of the activity//^r atio as described by Branzoli & Massey (1974a). Using the ratio determined by them for fully functional enzyme, we found that the AO preparation used in this work contained 30% functional Mo centers.…”

“…The functional Mo center of AO almost certainly contains a terminal sulfur ligand since it can be reacted with cyanide to yield thiocyanate (Branzoli & Massey, 1974a) and a "desulfo" Mo species with EPR properties (in the Mov oxidation state) and reduction potentials similar to those described for the xanthine oxidizing enzymes (Bray, 1980a,b; Table II) which are known to contain such a Mo=S structure in the functional but not in the cyanide-treated enzyme (Bordas et al, 1980;Cramer et al, 1981). Adducts of the Mov center with ethylene glycol and formyl groups (derived upon reaction with formaldehyde or methanol; Bray, 1980a) yield similar EPR spectra in AO and XO.…”

“…Desulfo AO was prepared as described by Branzoli & Massey (1974a). The formaldehyde, methanol, and desulfo ethylene glycol adducts of AO were prepared by procedures previously described for the formation of the analogous adducts in XO (Pick et al, 1971;Lowe et al, 1976).…”

“…In its functional form, the AO Mo center contains a cyanolyzable sulfur atom (Branzoli & Massey, 1974a) which EX-AFS (Bordas et al, 1980;Cramer et al, 1981) and EPR studies (Malthouse & Bray, 1980) have shown to be due to 1 Abbreviations: AO, rabbit liver aldehyde oxidase; Fe/S I, ironsulfur center with gav < 2.0 in the reduced enzyme; Fe/S II, iron-sulfur center with gav > 2.0 in the reduced enzyme; XO, milk xanthine oxidase; XDH, avian liver xanthine dehydrogenase; EXAFS, X-ray absorption fine structure, extended; EDTA, ethylenediaminetetraacetic acid. a Mo=S structure in XO and XDH.…”