INVOLVEMENT OF A FLAVIN IMINOQUINONE METHIDE IN THE FORMATION OF 6-HYDROXYFLAVIN MONONUCLEOTIDE IN TRIMETHYLAMINE DEHYDROGENASE - A RATIONALE FOR THE EXISTENCE OF 8-ALPHA-METHYL AND C6-LINKED COVALENT FLAVOPROTEINS

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, an d Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the a ctive site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and i nactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of sa mples of the native and recombinant wild-type trimethylamine dehydroge nases also revealed the presence of B-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of F MN in trimethylamine dehydrogenase that involves an electrophilic flav in iminoquinone methide. The proposed mechanism involving the flavin i minoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enz ymes via the 8 alpha position, thus providing a rationale for the evol ution of covalent flavoproteins in general. Covalent linkage at C6 or the 8 alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone meth ide, respectively.