No completely satisfying explanation has been provided for the first question. The attachment of an aminoacyl group to the 8␣-or 6-position of riboflavin does not confer any unusual properties on the flavin, either free in solution or in an enzyme, with the exception of the ultraviolet-visible spectrum of 6-Scysteinylriboflavin, which is quite different from that of other forms of free and bound aminoacyl flavins (2, 3). While the oxidation-reduction potentials (E m 7 ) are about 50 -60 mV more positive for aminoacyl flavins than for unmodified forms (2), this increase in potential can also be achieved by noncovalent interactions with protein. Additionally, enzymes with covalently bound flavins do not catalyze a unique or specific set of reactions.As to the second question, it is known that 2-electron reduction of protein-free 8␣-O-tyrosylriboflavin or 8␣-S-cysteinylsulfonylriboflavin cause expulsion of the aminoacyl groups, thus producing unmodified, oxidized riboflavin (2, 4). The principle of microscopic reversibility suggests that the reverse reaction could occur within an enzyme, with or without intervention of an external enzyme. A mechanism for covalent flavinylation, which has long been in the literature, is shown in Fig. 2 (5, 6). An analogous mechanism was proposed for nonenzymic basecatalyzed nucleophilic attack at the 8␣-carbon of riboflavin derivatives in organic solvents (7).It has been suggested that covalent tethering might require prior activation of the flavin or proteins, e.g. a high energy phosphate bond (8). Of several enzymes studied to date, no specific enzyme has been implicated in the covalent modification process, which contrasts with examples of nonflavin-cofactor covalent attachment to apoenzymes (9 -13).The structural genes of several bacterial enzymes containing covalently bound flavin have been cloned into vectors for expression in new hosts: succinate dehydrogenase, fumarate reductase (14, 15), sarcosine oxidase (16), 6-hydroxy-D-nicotine oxidase (6-HDNO) 1 from Arthrobacter aerogenes (15) (all containing 8␣-N 3 -histidyl-FAD), trimethylamine dehydrogenase

The bifunctional enzyme, FAD synthetase (FS), from Corynebacterium ammoniagenes was overproduced in Escherichia coli and purified, and its steady-state kinetic properties were investigated. Although FMN is an intermediate product in the conversion of riboflavin to FAD, FMN must be released after formation, and then rebind for adenylylation. It was shown that adenylylation of FMN is reversible; FAD and pyrophosphate can be converted to FMN and ATP by the enzyme. In contrast, under the conditions studied, phosphorylation of riboflavin is irreversible. A method is described for analysis of two catalytic cycles, occurring on one enzyme, which have a substrate and/or product in common. The binding order for the phosphorylation cycle of FS was established as riboflavin(in), ATP(in), ADP(out), and FMN(out). The order for the adenylylation cycle was ATP(in), FMN(in), pyrophosphate(out), and FAD(out). A set of steady-state constants was determined, and without additional optimization, these constants were sufficient to describe experimental progress curves for conversion of riboflavin to FAD. In independent studies, it was demonstrated that FMN binds to apo-FS with a dissociation constant of 6-7 microM, which is 2 orders of magnitude higher than the KD value for riboflavin. For the steady-state kinetic analysis, this represents reversible binding of FMN(out) in the phosphorylation cycle (cycle I), which effectively inhibits catalysis in the adenylylation cycle (cycle II).

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.