Autolysates of brewer's yeast contain several isoenzymes of fumarate reductase. One group (Type I) has a molecular weight of 62,000 to 63,000, as determined by chromatography on Sephadex, and 5 components in the group have been detected and partially or completely separated on triethylaminoethyl cellulose. Another group (Type II) consists of at least 2 isoenzymes. The predominant component shows a molecular weight of 34,000 and the minor one 112,000 ± 10,000 on Sephadex. One of the Type I enzymes has been extensively purified until it shows a single component on gradient centrifugation, although it appears to be heterogeneous on polyacrylamide electrophoresis. The prosthetic group is FAD and, in addition, iron and possibly, copper appear to be present. In contrast to mitochondrial succinate dehydrogenase the FAD is not covalently bound and the non heme iron is not linked to labile sulfide. Preliminary electron paramagnetic resonance data suggest that the iron may undergo oxidation‐reduction during the catalytic cycle. The absorption spectrum manifests the 450 mμ band of flavoproteins but there is an anomalous absorption below 400 mμ and in the long wave‐length region. Dithionite and succinate bleach the 450 mμ band and fumarate partially restores the color of the enzyme.
The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114,000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58,000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T. P., & Hopper, D. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4626-4630], however, indicate an alpha 2 beta 2 structure and a much lower molecular mass (approximately 8000) for the cytochrome subunit. In this paper we report data confirming the conclusions of X-ray crystallographic analysis. From quantitative amino acid analysis, the molecular mass of the flavoprotein monomer is shown to be 48,600 +/- 2200 and that of the cytochrome 8780 +/- 250. These values have been confirmed by gel electrophoresis under denaturing conditions. Gel chromatography under nondenaturing conditions shows that the isolated flavoprotein exists as a dimer, whereas the isolated cytochrome is a monomer. The complete amino acid sequence of the cytochrome c subunit is presented and is shown to have regions of homology to other bacterial c-type cytochromes. The partial N-terminal amino acid sequence (56 amino acids) of the flavoprotein subunit is also reported. The implications of the now established tetrameric structure of the flavocytochrome on data in the literature regarding the redox and association properties of the subunits are examined.
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