Presence of a flavin semiquinone in methanol oxidase.

Abstract

Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site. A methanol oxidase has been isolated from the yeast Hansenula polymorpha DL-1 (1). The enzyme contains FAD and is an octamer with a subunit molecular weight of 83,000 (1). The published spectrum of a similar enzyme attracted our attention because it indicates that a chromophore other than FAD is present (2). The spectrum suggests that the chromophore might be the semiquinone of FAD. We, therefore, undertook experiments to fully characterize the additional chromophore and to identify its role in the catalytic process. METHODS Enzyme Isolation and Assay. H. polymorpha strain DL-1 was a gift of Cooney. The enzyme was isolated as described (1, 3), and it was homogeneous upon NaDodSO4 gel electrophoresis. Its specific activity was 17 international units/mg. Enzyme activity was determined at 37'C by measuring 02 consumption in an 02 electrode. The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.5), 0.3 AM catalase, 6.5 mM methanol, and 0.1 ,uM enzyme.Analytical Procedures. All optical spectra were determined with a Perkin Elmer model 559 spectrophotometer. in which 1/T1 is the total relaxation rate, 1/T? is the diamagnetic control, and [R.] is the concentration of flavin semiquinone determined by double integration of the ESR spectra by using a picrylhydrazyl standard. The diamagnetic controls were the buffer (0.3 M potassium phosphate, pH 7.5) containing the same amount (7.6 mg/ml) of the diamagnetic protein, pyruvate kiThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. Fig. 1. The ratio of absorbances at 375 nm and 460 nm suggests that a flavin semiquinone is present in addition to oxidized flavin. From the spectrum of the oxidized and reduced enzyme, the nature and number of flavin species present were determined. The results are summarized in Table 1. There are five flavin semiquinones per octamer and two oxidized flavins that can be reduced upon substrate addition. Addition of Na2S204 or of the suicide inactivator cyclopropanol (unpublished results) also results in the reduction of the...

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