Studies of Aldehyde Oxidase by Electron Paramagnetic Resonance Spectroscopy: I. Spectra at Equilibrium States

Abstract

Abstract Aldehyde oxidase from rabbit liver was studied by electron paramagnetic resonance (EPR) spectroscopy. Reduction by substrate elicited three types of signals characteristic of free radicals, molybdenum(V), and a non-heme iron complex, respectively. The properties of these signals are described, and the representation of each species in the corresponding signal is quantitatively assessed. Under the specific conditions which yielded a maximum for each of the signal types, 24% of the flavin was in the semiquinone form (g = 2.00), 25% of the iron was in the reduced form responsible for the signal of g = 1.94, and 15 to 20% of the molybdenum was in the Mo(V) state with a signal at g = 1.97. Anaerobic titration of the enzyme with dithionite indicated that reduction of only 1 iron out of each 4 (per flavin) results in the appearance of the g = 1.94 signal, in agreement with the quantitative recovery in the EPR signal. When titration of the enzyme with N-methylnicotinamide was followed by EPR spectroscopy, partial formation of flavin semiquinone and accumulation of reducing equivalents in the non-heme iron complex was observed during consumption of the first 5 to 6 electrons per flavin. Extensive reduction of molybdenum ensued after an additional 1 to 2 eq. Spectrophotometry suggests that the enzymic components which accept electrons from substrate and are not detectable by EPR spectroscopy are that portion of the flavin which is fully reduced and the remainder of the non-heme iron. Rapid titrations, insufficient to permit equilibration between enzyme and substrate, showed migration of reducing equivalents from molybdenum toward flavin and non-heme iron with increasing time. These experiments show that intramolecular electron transfer can be effectively stopped at a low temperature. Measurements of electron spin relaxation of reduced enzyme suggest intramolecular magnetic interactions among the various paramagnetic species generated during the titrations.