Control of C4a-Hydroperoxyflavin Protonation in the Oxygenase Component of p-Hydroxyphenylacetate-3-hydroxylase

Abstract

The protonation status of the peroxide moiety in C4a-(hydro)peroxyflavin of p-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The pKa for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ± 0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency of these mutants is lower than that of the WT enzyme. Solvent kinetic isotope effect studies indicate that proton transfer is not the rate-limiting step in the formation of C4a-OOH. All data suggest that His396 may act as an instantaneous proton provider for the proton-coupled electron transfer that occurs before the transition state of C4a-OOH formation.

Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer

“…The pK a of C4aOOH in wild type C 2 (9.8±0.2) is higher than that of the H396V and H396A mutants (~7). 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 (1.95 Å) ( Figure 5) is also expected to assist H 2 O 2 elimination. Therefore, H 2 O 2 elimination in C 2 could be modulated by the interplay of the hydrogen bond interaction with Ser171 and Trp112.…”

“…The model proposed for the C 2 reaction is compatible with the absence of a solvent kinetic isotope effect (SKIE) for the formation of C4aOOH by this enzyme. 28 This implies that the proton transfer from His396 occurs rapidly before the formation of C4aOOH in C 2 and is not a rate-limiting step for the overall process of C4aOOH formation. The rate constants for the formation of C4aOOH formation in the H396N, H396A, and H396V variants were approximately 30-fold, 100-fold, and 300-fold slower than that of the wild-type enzyme, also agreeing with the catalytic role proposed for His396.…”

“…Recently, Chenprakhon et al 28 reported that His396 is important for maintaining a 21 protonated form and high pK a of C4aOOH. Their study suggested that His396 may be a proton source for PCET and that initially His396 should be fully-protonated before the oxygen reaction.…”

“…27a A recent investigation has shown that His396, which is located near the C4a of the isoalloxazine ring (the N ε of His396 is ~4.6 Å from the flavin C4a position), is important for maintaining the high pK a of the peroxide group of C4a-hydroperoxyflavin. 28 We speculated that this His residue could participate in a PCET mechanism similar to His548 in the reaction of P2O (the N ε of His548 in P 2 O is~4.5 Å from the C4a). 29 Therefore, in this work, the reaction of C 2 was used as a model to investigate the mechanism of oxygen activation in a flavin-dependent monooxygenase, in which the C4aOOH formation is rapid and robust.…”

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity

“…p-Hydroxyphenylacetate hydroxylase C 2 also contains its His-396 at a very similar position to His-388 in TdsC. Extensive kinetic studies on this enzyme suggested that His-396 acts as a proton source for the protoncoupled electron transfer before the transition state of C4ahydroperoxide formation and that this residue is important for maintaining C4a-hydroperoxyflavin in the protonated form (35,37). In the case of TdsC, the substitution of Tyr-93 with Phe resulted in no detectable activity for BT or indole oxygenation, indicating that the presence of the phenyl group in Y93F is not sufficient for the oxygenation activity of TdsC and that the hydroxy group of Tyr-93 plays a crucial role in the oxygenation of BT and indole.…”