JBC, Vol. 251, Issue 16, 4872-4881, Aug, 1976
Nucleophilic addition reactions of free and enzyme-bound deazaflavin
M. S. Jorns and L. B. Hersh
DeazaFMN-containing glycolate oxidase has been prepared and shown to
catalyze the stereospecific transfer of the alpha-hydrogen from substrate
to enzyme-bound deazaFMN. The reaction of sulfite, cyanide, and
hydroxylamine with several deazaflavin-containing enzymes (glycolate
oxidase, D-amino acid oxidase, glucose oxidase, N-methylglutamate
synthetase) and free deazaFMN has been examined. All the deazaflavin
systems tested form reversible 1:1 complexes with sulfite and cyanide. The
pH dependence of the reaction of free deazaFMN with cyanide indicates that
cyanide anion is the reacting nucleophile. Hydroxylamine complexes are
formed with deazaFMN glycolate oxidase and deazaFAD glucose oxidase. The
effectiveness of the various nucleophilic reagents in complex formation
decreases in the following order: sulfite greater than cyanide greater than
hydroxylamine. The relative stability observed for the sulfite and cyanide
complexes formed with various deazaflavin systems (glycolate oxidase
greater than D-amino acid oxidase greater than free deazaFMN) follows the
same trend observed for the stability of the sulfite complexes formed with
the corresponding flavin system. A correlation is also observed between the
reduction potential (E'o) of the deazaflavin system (glycolate oxidase (-
170 mV) greater than D-amino acid oxidase (-240 mV) greater than free
deazaFMN (-178 mV) and the stability of the deazaflavin-nucleophile
complexes. The following evidence indicates that deazaflavin systems are
generally more susceptible toward nucleophilic attack than corresponding
flavin system: (a) with the exception of glucose oxidase, the dissociation
constants for the deazaflavin-sulfite complexes are at least 1 order of
magnitude less than the corresponding flavin sulfite complexes; (b) the
least reactive nucleophile, hydroxylamine, does not form a complex with any
of the flavin systems. In the case of cyanide, a complex is formed only
with native glycolate oxidase, which is the flavin-containing system most
susceptible to attack by the more reactive sulfite. Formation of the
various (deaza)flavin-nucleophile complexes is characterized by a bleaching
of the longer wavelength absorption band of the chromophore and increases
in absorption below the isosbestic point of the reaction in the
near-ultraviolet region of the spectrum. These results are consistent with
the formation of covalent adducts via attack of the various nucleophiles at
position 5 of (deaza)flavin. The reaction with cyanide provides the first
example of a reversible addition of carbanion to enzyme-bound
(deaza)flavin.
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