6-Mercapto-FAD and 6-thiocyanato-FAD as active site probes of phenol hydroxylase.

Recently, the synthesis and properties of several 6-substituted flavins as active site probes for flavoproteins have been reported (Ghisla, S., Massey, V., and Yagi, K. (1986) Biochemistry 25, 3282-3289). Here, we report results of experiments in which 6-thiocyanato-FAD and 6-mercapto-FAD have been substituted for the native flavin of phenol hydroxylase. The 6-SCN-FAD enzyme was converted spontaneously to the 6-mercaptoflavin form probably due to dissociation of flavin, followed by attack of external protein thiols. The pK alpha values of uncomplexed and phenol-bound 6-mercapto-FAD enzyme were determined. Both the spontaneously formed 6-mercapto-FAD enzyme and the enzyme reconstituted with preformed 6-mercapto-FAD were treated with a variety of thiol-specific reagents, and reaction rates were followed by spectroscopic means. Comparison with the corresponding rates found with free flavin suggested a high degree of accessibility to the flavin 6-position. Accessibility was somewhat decreased in the presence of phenol. Upon treatment with low concentrations of methyl methanethiosulfonate or N-ethylmaleimide (NEM), extremely rapid spectral changes were apparent. The former reaction, however, was reversed spontaneously within 2 h. Reaction with NEM was biphasic, with spectral changes consistent with the mechanism previously proposed (Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824), followed by a small absorbance decrease due to protein conformational changes. The NEM reaction is unusual, being easily reversed by addition of excess dithiothreitol.

• Publication year

  1991

• Venue

  Journal of Biological Chemistry

• Publication date

  1991-05-05

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(18)92974-1PMID 2022645
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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