J. Biol. Chem., Vol. 265, Issue 4, 1952-1957, Feb, 1990

Absorption spectra of the hydroxycyclohexadienyl radicals of substrates for phenol hydroxylase

RF Anderson, KB Patel and MR Stratford
Gray Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom.

The absorption spectra of the hydroxycyclohexadienyl radicals formed upon the addition of OH radicals to six substrates for phenol hydroxylase have been determined using pulse radiolysis. Combining the radical spectra of thiophenol (lambda max, 390 nm; epsilon, 10,500 M-1 cm-1) and resorcinol (lambda max, 340 nm; epsilon, 4,100 M-1 cm-1) with their respective published spectra of enzyme-bound reduced flavin that is substituted in the C(4a) position of the dihydroflavin ring gave composite spectra that closely match the spectra formed concomitantly with the introduction of an oxygen atom into the substrates, the so- called Intermediate II species. A similar procedure for the substrates hydroquinone, 3-aminophenol, 3-chlorophenol, and 3-methylphenol yielded spectra that are also consistent with the known characteristics of their Intermediate II species. These spectral results give further support to the proposed biradical mechanism (Anderson, R.F., Patel, K. B., and Stratford, M. R. L. (1987) J. Biol. Chem. 262, 17475-17479) for the functioning of this class of flavoprotein hydroxylases.
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