Coordinated production and utilization of FADH2 by NAD(P)H - Flavin oxidoreductase and 4-hydroxyphenylacetate 3-monooxygenase

Abstract

4-Hydroxyphenylacetate (4HPA) 3-monooxygenase (HpaB) is a reduced flavin adenine dinucleotide (FADH2) utilizing monooxygenase. Its cosubstrate, FADH2, is supplied by HpaC, an NAD(P)H - flavin oxidoreductase. Because HpaB is the first enzyme for 4HPA metabolism, FADH2 production and utilization become a major metabolic event when Escherichia coli W grows on 4HPA. An important question is how FADH2 is produced and used, as FADH2 is unstable in the presence of free O2. One solution is metabolic channeling by forming a transitory HpaB - HpaC complex. However, our in vivo and in vitro data failed to support the interaction. Further investigation pointed to an alternative scheme for HpaB to sequester FADH2. The intracellular HpaB concentration was about 122, μM in 4HPA-growing cells, much higher than the total intracellular FAD concentration, and HpaB had a high affinity for FADH2 (Kd of 70 nM), suggesting that most FADH2 is bound to HpaB in vivo. The HpaB-bound FADH2 was either used to rapidly oxidize 4HPA or slowly oxidized by O2 to FAD and H2O2 in the absence of 4HPA. Thus, HpaB's high intracellular concentration, its high affinity for FADH2, its property of protecting bound FADH2 in the absence of 4HPA, and its ability to rapidly use FADH2 to oxidize 4HPA when 4HPA is available can coordinate FADH2 production and utilization by HpaB and HpaC in vivo. This type of coordination, in responding to demand, for production and utilization of labile metabolites has not been reported to date.