Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

Abstract

p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C 2 ), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ϳ30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H 2 O 2 elimination. Studies on pH dependence showed that the rate constant of H 2 O 2 elimination in H396N and H396V increased when pH increased with pK a >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK a >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H 2 O 2 elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H 2 O 2 elimination in S171A and S171T were ϳ1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H 2 O 2 elimination increased with pH rise and exhibited an approximate pK a of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C 2 .

Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

“…The C4a-hydroperoxy-FMN intermediates in both reactions have absorbance maxima at 390 nm (inset in supplemental Figs. 8 and 9), similar to those of wild-type C 2 and other variants (15,21,27).…”

“…The spectrum in the inset in Fig. 2A had a maximum absorption peak at 390 nm, similar to that of a C4a-hydroperoxy-FMN intermediate in the reactions of wild-type C 2 and other variants (15,21,27). Therefore, the first phase shown in Fig.…”

“…X-ray structures of the oxygenase components from A. baumannii at 2.3-2.8 Å resolution (23) and Thermus thermophilus HB8 at 1.3-2.0 Å resolution (24,25) and the reductase component from Sulfolobus tokodaii (26) at 1.7-2.3 Å resolution have been solved. Recent site-directed mutagenesis studies of the HPAH from A. baumannii have shown that Ser-171 is a key residue for stabilization of the reactive intermediate, C4a-hydroperoxy flavin, whereas His-396 facilitates intermediate formation (27). Although the ability of C 2 to prolong the lifetime of C4a-hydroperoxy flavin is necessary for substrate hydroxylation (27), the enzyme must have another fine-tuned mechanism for efficient oxygen transfer because between pH 6.0 and 10.0 the product conversion percentage is ϳ90% without wasteful H 2 O 2 production, and the rate constants for hydroxylation remain constant at 15-17 s Ϫ1 (15).…”

“…Recent site-directed mutagenesis studies of the HPAH from A. baumannii have shown that Ser-171 is a key residue for stabilization of the reactive intermediate, C4a-hydroperoxy flavin, whereas His-396 facilitates intermediate formation (27). Although the ability of C 2 to prolong the lifetime of C4a-hydroperoxy flavin is necessary for substrate hydroxylation (27), the enzyme must have another fine-tuned mechanism for efficient oxygen transfer because between pH 6.0 and 10.0 the product conversion percentage is ϳ90% without wasteful H 2 O 2 production, and the rate constants for hydroxylation remain constant at 15-17 s Ϫ1 (15). The pH independence of the C 2 reaction implies that the protonation status of key components required for oxygen atom transfer is unchanged in this pH range.…”

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

“…For Class B flavoprotein monooxygenases such as phenylacetone monooxygenase (31), cyclohexanone monooxgenase (32), and L-ornithine monooxygenase (33)(34)(35), the binding of NAD(P) ϩ is required to stabilize the C4a-peroxyflavin intermediate (36,37). For Class C enzymes such as bacterial luciferase (38,39) and Class D enzymes such as the oxygenase component of p-hydroxyphenylacetate hydroxylase (27,40), C4a-hydroperoxyflavin can be detected in the absence of any bound ligand.…”