Altered Balance of Half-reactions in p-Hydroxybenzoate Hydroxylase Caused by Substituting the 2′-Carbon of FAD with Fluorine

Abstract

Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2-fluoro-2-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2-center of the ribityl chain was replaced with fluorine in an inverted configuration. The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate. In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound. Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB. Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158). Thus, whereas the change that induces the out conformation is detrimental to the oxidative halfreaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis.

Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen

“…3B). This is consistent with the formation of flavin hydroperoxide, which typically exhibits a 10–15 nm blue shift [35,36]. This blue shift takes place in the red to the cyan transition ( k CD in Fig.…”

l‐Lactate dehydrogenation in flavocytochrome b2

“…1), revealing a role for the 2′‐OH position of the flavin ribityl side chain in maintaining the correct geometry of a key active site residue. Interestingly, only a few studies have so far explored the role of the flavin side chain in flavoenzymes [25–29] and our results support its importance also in Fcb2.…”

Deflavination and reconstitution of flavoproteins

“…Flavin analogs modified in the ribityl side chain may also provide insight into flavoprotein function and mechanism [54,84–87]. For medium‐chain acyl‐CoA dehydrogenase, it was shown that the replacement of natural FAD by 2′‐deoxy‐FAD reduces the activity of the enzyme about a millionfold [84].…”