The possibility that the spectrum of intermediate two, seen in the course of reaction of flavoenzyme phenol hydroxylases, may be attributable to iminol isomers of a flavin-derived 6-arylamino-5-oxo(3H,5H)uracil.

Abstract

The possibility that the spectrum of intermediate two, seen in the course of reaction of flavoenzyme phenol hydroxylases, may be attributable to iminol isomers of a flavin-derived 6-arylamino-5-oxo(3H,5H)uracil (flavin mixed-function oxidase/hydroxylation) ALBERT WESSIAK, J. BARRY NOAR, AND THOMAS C. BRUICE* Department of Chemistry, University of California, Santa Barbara, CA 93106 Contributed by Thomas C. Bruice, September 19, 1983 ABSTRACT A commonly held view of the mechanism of flavin mixed-function oxidases is that enzyme-bound 4a-hydroperoxyflavin (4a-FIHOOH) undergoes ring opening to provide a carbonyl oxide (IV), which, after transferring an oxene equivalent to substrate, yields a 6-arylamino-5-oxo(3H,5H)-uracil (I). The latter is then thought to undergo ring closure to form a 4a-hydroxyflavin (4a-FIHOH), which by loss of water yields flavin (scheme I). A close structural analogue of I (i.e., III) has been synthesized. Comparison of the spectra of III (and II), taken in solvents of widely differing dielectric constants and in a strongly basic medium, with those of the intermediate(s) observed to be formed in time between 4a-FIHOOH and 4a-FIHOH has shown that the enzyme-bound intermediate(s) does not resemble spectrally I nor its iminol tautomers.

Use of riboflavin-binding protein to investigate steric and electronic relationships in flavin analogs and models.

“…Compounds II were synthesized as models for an intermediate that appears between the hydroperoxy and hydroxy species during the hydroxylation reaction catalyzed by flavin-dependent phenolic hydroxylases (17). This intermediate has been postulated to be a 4a,5-ring-opened species (17) in which N(5) is substituted by two hydrogens instead of the methyl groups, as in compounds H (14)(15)(16). Steric aspects of a possible 4a,5-ring-opening mechanism have up to now not been considered explicitly.…”

“…If the absorbance spectra of N(5)-alkylated models such as Ha and Ilb are good approximations to the enzyme-bound case [in which N(5) bears two hydrogens], then the ring-opening mechanism of hydroxylation seems to be wrong, since the absorbance spectra of ha and IIb are quite different from the spectrum of the enzyme-bound intermediate (14)(15)(16)). An alternative interpretation of the results would be that compounds H have such severe steric constraints, due to the methyl groups on N(5), as to be unsuitable models.…”

“…The 4a,5-ringopened isoalloxazines (6-{[2-(dimethylamino)-4,5-dimethylphenyl]methylamino}-5-oxo-3H,5H-uracils) Ha (R3 = CH3) and lIb (R3 = H) were synthesized (14)(15)(16) to compare their spectral properties to those of enzyme-bound intermediates, observed during the hydroxylation reaction of flavindependent phenolic hydroxylases (17). Pyrimidopteridines (2,3,4,6,7,8,9 -heptahydro -2,4,6,8 -tetraoxopyrimido -5, -4g-pteridines), III (R3 or R7 = H or CH3), were designed to provide flavin cofactor analogs with more negative redox potentials than the native FMN of FAD (18).…”

“…The UV/visible spectra of the compounds II free in solution and those of enzyme-bound intermediates turned out to be quite dissimilar (14)(15)(16). It therefore became of interest to determine whether the effect of apoprotein interactions on the physical properties of compounds II might account for the differences.…”

Theoretical investigation of the [1,2]-sigmatropic hydrogen migration in the mechanism of oxidation of 2-aminobenzoyl-CoA by 2-aminobenzoyl-CoA monooxygenase/reductase

“…It is generally accepted that flavin-4a-hydroperoxides form in the active sites of flavindependent mixed-function oxidases when reduced flavin reacts with molecular oxygen (6)(7)(8)(9)(10)(11)(12). It was determined that the monooxygen transfer involving 4a-hydroperoxyflavins does not occur from a carbonyl oxide formed on ring opening of the isoalloxazine ring between C4 and C4a (13,14), as originally proposed (1), but by nucleophilic attack on the terminal oxygen of the flavin 4a-hydroperoxide (15). Dioxygen transfer from the oxygen anion 4a-FlEt-OO Ϫ is known in model reactions (3,(16)(17)(18)(19)(20), but there is no evidence for the existence of a flavin dioxygenase enzyme.…”