Amino acid and sequence analysis of the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase

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Abstract
The flavocytochrome p-cresol methylhydroxylase from Pseudomonas putida has been reported to have a Mr of 114000 and to consist of two subunits, a flavoprotein and a cytochrome c, each with a Mr of 58000. Recent X-ray crystallographic data from our laboratories [Shamala, N., Lim, L. W., Mathews, F. S., McIntire, W., Singer, T.P., and 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 (.apprx. 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 48600 .+-. 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 of 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.