Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of aldehyde oxidase

Abstract

Molybdenum(V) e.p.r. spectra from reduced forms of aldehyde oxidase were obtained and compared with those from xanthine oxidase. Inhibited and Desulpho Inhibited signals from aldehyde oxidase were fully characterized, and parameters were obtained with the help of computer simulations. These differ slightly but significantly from the corresponding parameters for the xanthine oxidase signals. Rapid type 1 and type 2 and Slow signals were obtained from aldehyde oxidase, but were not fully characterized. From the general similarities of the signals from the two enzymes, it is concluded that the ligands of molybdenum must be identical and that the overall co-ordination geometries must be closely similar in the enzymes. The striking differences in substrate specificity must relate primarily to structural differences in a part of the active centre concerned with substrate binding and not involving the catalytically important molybdenum site.

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. The implication is that the second proton of both signal-giving species is bound at the same site in the two signal-giving species, with the difference between them involving differences in coordination geometry and/or orientation of substrate with respect to the molybdenum center .…”

“…Mo(V) signals corresponding closely to the “arsenite-inhibited” and “slow” signals seen with xanthine oxidase are also observed with aldehyde oxidase . Neither the “very rapid” or “rapid type 1” Mo(V) signals have been unambiguously documented with aldehyde oxidase using N -methylnicotinamide as substrate, however, although a “rapid type 1” signal is observed when 2-methylquinonine is used. , …”

The strict molybdate‐dependence of glucose‐degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme – an aldehyde oxidoreductase

“…EPR investigation of the aerobically purified protein revealed the so‐called ‘ desulpho‐inhibited ’ molybdenum‐cluster (Fig. 6a) as also found in nonfunctional forms of xanthine oxidase and aldehyde oxidase [32]. The g ‐values[32a] ( g xyz = 1.96, 1.97, 1.98, g av = 1.97) were in good agreement with those reported for the aldehyde oxidase from rabbit liver [33], although our samples were not treated with dithionite nor with ethylene glycol, indicating that any kind of modification had already occurred during the purification procedure.…”

The Mononuclear Molybdenum Enzymes

“…Notably, however, aldehyde oxidase has not been shown to manifest a “very rapid” type of EPR signal, and preferentially manifests the “rapid Type 2” signal (with coupling to two equivalent protons) rather than the “rapid Type 1” (with coupling to two inequivalent protons) more commonly seen with wild-type enzyme. 182d,206g,h,207 Genes encoding aldehyde oxidases from cow, 208 human, 209 rat, 210 and mouse 211 have all been cloned and shown to share similar intron/exon structures, a reflection of their close evolutionary relationship. In humans, in addition to the single Aox1 gene, 209 there are two pseudogenes that are not expressed and represent vestigial remnants of two of the three additional isoforms seen in mouse.…”