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

Abstract

Rabbit liver aldehyde oxidase (AO), like milk xanthine oxidase (XO) and chicken liver xanthine dehydrogenase (XDH), possesses the following prosthetic groups: FAD, a functional Mo center, and 2 spectroscopically distinct Fe-S centers, one with gav < 2.0 (termed Fe/S I) and the other with gav > 2.0 (termed Fe/S II) in the reduced enzyme. EPR spectra for the MoV species were nearly identical in AO and XO for a number of enzyme complexes, and the midpoint reduction potentials for functional MoVI/MoV (-359 mV) and MoV/MoIV (-351 mV) were nearly the same in all 3 enzymes (50 mM phosphate, pH 7.8). A strong magnetic interaction between MoV and reduced Fe/S I, previously detected in XO and XDH, was also found in AO. No MoV-Fe/S II interaction could be detected in AO (nor in XO). In contrast, the order of reduction of Fe/S I and Fe/S II, as measured from their midpoint potentials, is reversed in AO (Em = -207 and -310 mV, respectively), as compared to XO (Em = -280 and -245 mV, respectively), in phosphate buffer at pH 7.8. The oxidized-reduced extinction coefficients at 450 and 550 nm for the 2 centers are also apparently reversed in AO and XO. Although magnetic interaction between FAD and one or both reduced Fe/S centers has been detected in both AO and XO, no magnetic interaction between the 2 reduced Fe/S centers themselves was found in AO (although such interaction has been seen in XO). The average FAD reduction potential is substantially more positive in AO (Em for FAD/FADH.cntdot., -258 mV; FADH.cntdot./FADH2, -212 mV at pH 7.8) than in XO or XDH. Although the properties and immediate environment of the functional Mo center are conserved in the 3 Mo hydroxylase enzymes, and all 3 enzymes possess the same set of prosthetic groups, the properties of the groups which transfer electrons from the Mo to the ultimate electron acceptor can vary substantially in AO, XO, and XDH.