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

  • Kittisak Thotsaporn
    Footnotes
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,
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  • Pirom Chenprakhon
    Footnotes
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,
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  • Jeerus Sucharitakul
    Footnotes
    Affiliations
    Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand, and
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  • Andrea Mattevi
    Affiliations
    Department of Genetics and Microbiology, University of Pavia, 27100 Pavia, Italy
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  • Pimchai Chaiyen
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: 662-2015596; Fax: 662-3547174
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand,
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  • Author Footnotes
    * This work was supported by the Thailand Research Fund Grant BRG5480001 and a grant from the Faculty of Science, Mahidol University (to P. C.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. S1–S3.
    1 Supported by the Royal Golden Jubilee Ph.D. Program Grant PHD/0008/2549.
    2 Supported by the Institute for the Promotion of Teaching Science and Technology.
    3 Supported by the Thailand Research Fund Grant MRG5380240.
Open AccessPublished:June 16, 2011DOI:https://doi.org/10.1074/jbc.M111.241836
      p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C2), 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 ∼30-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 H2O2 elimination. Studies on pH dependence showed that the rate constant of H2O2 elimination in H396N and H396V increased when pH increased with pKa >9.6 and >9.7, respectively, similar to the wild-type enzyme (pKa >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H2O2 elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H2O2 elimination in S171A and S171T were ∼1400-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 H2O2 elimination increased with pH rise and exhibited an approximate pKa 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 C2.

      Introduction

      Flavin-dependent monooxygenases incorporate one atom of molecular oxygen into their organic substrates and are involved in a wide variety of biological redox reactions. The enzymes can be classified into six subclasses according to their reactions and amino acid sequences or into two major types according to polypeptide components involved in the reaction (
      • van Berkel W.J.
      • Kamerbeek N.M.
      • Fraaije M.W.
      ,
      • Fagan R.L.
      • Palfey B.A.
      ). Single-component flavoprotein monooxygenases catalyze flavin reduction and substrate oxygenation using single polypeptides, which have been studied for many decades (
      • Fagan R.L.
      • Palfey B.A.
      ,
      • Ballou D.P.
      • Entsch B.
      • Cole L.J.
      ). Only in the past decade have two-component monooxygenases received more attention (
      • van Berkel W.J.
      • Kamerbeek N.M.
      • Fraaije M.W.
      ,
      • Fagan R.L.
      • Palfey B.A.
      ,
      • Ballou D.P.
      • Entsch B.
      • Cole L.J.
      ,
      • Ellis H.R.
      ). They have been found to catalyze flavin reduction and substrate oxidation using separate polypeptides and are important for the metabolism of aromatic and aliphatic compounds in bacteria (
      • Ellis H.R.
      ,
      • Chaiyen P.
      • Suadee C.
      • Wilairat P.
      ,
      • Kirchner U.
      • Westphal A.H.
      • Müller R.
      • van Berkel W.J.
      ,
      • Otto K.
      • Hofstetter K.
      • Röthlisberger M.
      • Witholt B.
      • Schmid A.
      ); biosynthetic pathways of antibiotics and cancer drugs, such as actinorhodin (
      • Valton J.
      • Mathevon C.
      • Fontecave M.
      • Nivière V.
      • Ballou D.P.
      ), rebeccamycin (
      • Yeh E.
      • Cole L.J.
      • Barr E.W.
      • Bollinger Jr., J.M.
      • Ballou D.P.
      • Walsh C.T.
      ), violacein (
      • Balibar C.J.
      • Walsh C.T.
      ), enediyne (
      • Lin S.
      • Van Lanen S.G.
      • Shen B.
      ), angucycline (
      • Koskiniemi H.
      • Metsä-Ketelä M.
      • Dobritzsch D.
      • Kallio P.
      • Korhonen H.
      • Mäntsälä P.
      • Schneider G.
      • Niemi J.
      ), kijanimicin (
      • Zhang H.
      • White-Phillip J.A.
      • Melançon 3rd, C.E.
      • Kwon H.J.
      • Yu W.L.
      • Liu H.W.
      ), and kutzneride (
      • Heemstra Jr., J.R.
      • Walsh C.T.
      ); and bacterial pathogenesis (
      • Dresen C.
      • Lin L.Y.
      • D'Angelo I.
      • Tocheva E.I.
      • Strynadka N.
      • Eltis L.D.
      ). During the past few years, several crystal structures of the oxygenase components of these enzymes have been reported, including HpaB from Thermus thermophilus HB8 (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ), LadA from Geobacillus thermodenitrificans NG80–2 (
      • Li L.
      • Liu X.
      • Yang W.
      • Xu F.
      • Wang W.
      • Feng L.
      • Bartlam M.
      • Wang L.
      • Rao Z.
      ), RebH from Lechevalieria aerocolonigenes (
      • Yeh E.
      • Blasiak L.C.
      • Koglin A.
      • Drennan C.L.
      • Walsh C.T.
      ,
      • Bitto E.
      • Huang Y.
      • Bingman C.A.
      • Singh S.
      • Thorson J.S.
      • Phillips Jr., G.N.
      ), TftD from Burkholderia cepacia AC1100 (
      • Webb B.N.
      • Ballinger J.W.
      • Kim E.
      • Belchik S.M.
      • Lam K.S.
      • Youn B.
      • Nissen M.S.
      • Xun L.
      • Kang C.
      ), SMOA from Pseudomonas putida S12 (
      • Ukaegbu U.E.
      • Kantz A.
      • Beaton M.
      • Gassner G.T.
      • Rosenzweig A.C.
      ), HsaA from Mycobacterium tuberculosis (
      • Dresen C.
      • Lin L.Y.
      • D'Angelo I.
      • Tocheva E.I.
      • Strynadka N.
      • Eltis L.D.
      ), KijD3 from Actinomadura kijaniata (
      • Bruender N.A.
      • Thoden J.B.
      • Holden H.M.
      ), and ORF36 from Micromonospora carbonacea var. africana (
      • Vey J.L.
      • Al-Mestarihi A.
      • Hu Y.
      • Funk M.A.
      • Bachmann B.O.
      • Iverson T.M.
      ). Although many x-ray structures of these enzymes are known, only a few enzymes have been investigated for their reaction kinetics, and little is known about the functional roles of residues surrounding the flavin-binding site.
      All types of flavin-dependent monooxygenases use a reactive flavin-oxygen adduct intermediate, C4a-hydroperoxyflavin, for oxygenating various compounds (
      • van Berkel W.J.
      • Kamerbeek N.M.
      • Fraaije M.W.
      ,
      • Fagan R.L.
      • Palfey B.A.
      ,
      • Ballou D.P.
      • Entsch B.
      • Cole L.J.
      ). In the absence of substrates or under specific conditions, C4a-hydroperoxyflavin eliminates H2O2 to yield oxidized flavin (Fig. 1) without catalyzing the oxygenation. The degree of C4a-hydroperoxyflavin stability in the absence of a substrate varies among different enzymes ranging from rather long half-lives (e.g. bacterial luciferase (t½ = ∼350 s at 4 °C, pH 8.0) (
      • Suadee C.
      • Nijvipakul S.
      • Svasti J.
      • Entsch B.
      • Ballou D.P.
      • Chaiyen P.
      ), cyclohexanone monooxygenase (t½ = ∼300 s at 4 °C, pH 7.2) (
      • Sheng D.
      • Ballou D.P.
      • Massey V.
      ), oxygenase in the biosynthesis pathway of actinorhodin (t½ = ∼1400 s at 4 °C, pH 7.4) (
      • Valton J.
      • Mathevon C.
      • Fontecave M.
      • Nivière V.
      • Ballou D.P.
      ), and RebH halogenase (t½ = ∼63 h at 4 °C, pH 7.5) (
      • Yeh E.
      • Blasiak L.C.
      • Koglin A.
      • Drennan C.L.
      • Walsh C.T.
      )) to short half-life intermediates, where the C4a-hydroperoxyflavin is not kinetically stabilized unless a substrate is present (e.g. p-hydroxybenzoate hydroxylase (
      • Entsch B.
      • Cole L.J.
      • Ballou D.P.
      ,
      • Palfey B.
      • Ballou D.P.
      • Massey V.
      ) or 2-methyl-3-hydroxypyridine-5-carboxylic acid monooxygenase (
      • Chaiyen P.
      ,
      • Chaiyen P.
      • Brissette P.
      • Ballou D.P.
      • Massey V.
      ,
      • Chaiyen P.
      • Brissette P.
      • Ballou D.P.
      • Massey V.
      )). Currently, the factors that control the intermediate stability of various monooxygenases are unknown.
      Figure thumbnail gr1
      FIGURE 1Reaction of a two-component flavin-dependent monooxygenase in the absence of substrate. The first step of the reaction is the formation of a C4a-hydroperoxyflavin intermediate, and the second step is H2O2 elimination to form the oxidized flavin.
      The two-component flavin-dependent monooxygenase, p-hydroxyphenylacetate (HPA)
      The abbreviations used are: HPA
      p-hydroxyphenylacetate
      HPAH
      p-hydroxyphenylacetate hydroxylase 3-hydroxylase
      DHPA
      3,4-dihydroxyphenylacetate.
      3-hydroxylase (HPAH), has well understood reaction kinetics and its intermediate, C4a-hydroperoxy-FMN, has been found to be quite stable (t½ = ∼230 s for the enzyme from Acinetobacter baumannii and t½ = ∼140 s for the enzyme from Pseudomonas aeruginosa) (
      • Ruangchan N.
      • Tongsook C.
      • Sucharitakul J.
      • Chaiyen P.
      ,
      • Sucharitakul J.
      • Chaiyen P.
      • Entsch B.
      • Ballou D.P.
      ,
      • Chakraborty S.
      • Ortiz-Maldonado M.
      • Entsch B.
      • Ballou D.P.
      ). The enzyme catalyzes the hydroxylation of HPA to 3,4-dihydroxyphenylacetate (DHPA) (
      • Chaiyen P.
      • Suadee C.
      • Wilairat P.
      ). This enzyme has been identified from various bacterial species and can be grouped into two types based on sequence homology (
      • Thotsaporn K.
      • Sucharitakul J.
      • Wongratana J.
      • Suadee C.
      • Chaiyen P.
      ). The first type includes HPAHs from P. aeruginosa (
      • Chakraborty S.
      • Ortiz-Maldonado M.
      • Entsch B.
      • Ballou D.P.
      ), Escherichia coli (
      • Galán B.
      • Díaz E.
      • Prieto M.A.
      • García J.L.
      ), T. thermophilus HB8 (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ,
      • Kim S.H.
      • Hisano T.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ), and Sulfolobus tokodaii strain 7 (
      • Okai M.
      • Kudo N.
      • Lee W.C.
      • Kamo M.
      • Nagata K.
      • Tanokura M.
      ). These enzymes consist of a small reductase (16–20 kDa) and a large oxygenase component (54–59 kDa). A substrate of the oxygenase component (HPA) shows no effect on the flavin reduction, which occurs on the reductase component (
      • Chakraborty S.
      • Ortiz-Maldonado M.
      • Entsch B.
      • Ballou D.P.
      ). The other type of enzyme is HPAH from A. baumannii (
      • Chaiyen P.
      • Suadee C.
      • Wilairat P.
      ,
      • Thotsaporn K.
      • Sucharitakul J.
      • Wongratana J.
      • Suadee C.
      • Chaiyen P.
      ), which consists of a larger size reductase (C1) (35.5 kDa), compared with the previously mentioned reductases, and an oxygenase (C2) (47 kDa). In this type of HPAH, HPA binds to the reductase component and acts as an effector, which stimulates the flavin reduction (
      • Sucharitakul J.
      • Chaiyen P.
      • Entsch B.
      • Ballou D.P.
      ), as well as the reduced flavin transfer process from the reductase to the oxygenase (
      • Sucharitakul J.
      • Phongsak T.
      • Entsch B.
      • Svasti J.
      • Chaiyen P.
      • Ballou D.P.
      ). A structural comparison of C2 (
      • Alfieri A.
      • Fersini F.
      • Ruangchan N.
      • Prongjit M.
      • Chaiyen P.
      • Mattevi A.
      ) and the oxygenase component of HPAH from T. thermophilus HB8 (HpaB) (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ) has indicated that the overall folding of both enzymes belong to the folding of the acyl-CoA dehydrogenase superfamily. However, residues surrounding the flavin-binding site in both enzymes are different. Within a 5-Å distance from the flavin C4a and N5 loci, Ser-171 (2.9 Å from the flavin N5) and His-396 (4.5 Å from the flavin C4a) are present in C2 (
      • Alfieri A.
      • Fersini F.
      • Ruangchan N.
      • Prongjit M.
      • Chaiyen P.
      • Mattevi A.
      ) (Fig. 2), whereas Thr-185 and Arg-100 are present in HpaB (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ), with a similar distance but a slightly different geometry. The location of these residues at these strategic positions implies that they should be important for enzyme catalysis.
      Figure thumbnail gr2
      FIGURE 2The active site structures of C2 from A. baumannii. The crystal structure of the C2-FMNH complex (Protein Data Bank code 2JBS) and amino acid residues ∼5 Å from the flavin (yellow) C4a and N5 loci are shown. For clarity, side chains of residues 120–127 are not shown.
      The rate constant for H2O2 elimination in the C2 wild type increases upon an increase in pH and is associated with a pKa > 9.4; one of the speculations about this pKa is that it may belong to His-396, which may act as a general base to assist in the deprotonation of the N5-H to generate H2O2 (step 2 in Fig. 1) (
      • Ruangchan N.
      • Tongsook C.
      • Sucharitakul J.
      • Chaiyen P.
      ,
      • Alfieri A.
      • Fersini F.
      • Ruangchan N.
      • Prongjit M.
      • Chaiyen P.
      • Mattevi A.
      ). Alternatively, this pKa may reflect the pKa of the overall H2O2 elimination process, without the involvement of His-396 (
      • Ruangchan N.
      • Tongsook C.
      • Sucharitakul J.
      • Chaiyen P.
      ). Recently, a study on pyranose 2-oxidase, a flavin oxidase that stabilizes C4a-hydroperoxyflavin as a reaction intermediate, has shown that the N5-H bond breakage controls the overall process of the H2O2 elimination (
      • Sucharitakul J.
      • Wongnate T.
      • Chaiyen P.
      ). Therefore, we investigated the role of His-396 and Ser-171 in the formation and stabilization of the C4a-hydroperoxy-FMN in C2. Our findings indicated that His-396 was important for C4a-hydroperoxy-FMN formation and Ser-171 was important for the stabilization of the C4a-hydroperoxy-FMN.

      DISCUSSION

      Our findings here have shown that His-396 and Ser-171 are important for the formation and stabilization, respectively, of C4a-hydroperoxy-FMN in C2. Residues similar to His-396 and Ser-171 of C2 were also identified in the active sites of other two-component monooxygenases that are homologous to C2. Based on the structure of the oxygenase component (HpaB) of HPAH from T. thermophilus, the Oγ of Thr-185 is 2.8 Å from the flavin N5 and the guanidyl N of Arg-100 is 4.80 Å from the flavin C4a locus (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ). For the oxygenase component (TftD) of chlorophenol 4-monooxygenase from B. cepacia AC1100, Thr-192 is located ∼ 3 Å away from the flavin N5 (
      • Webb B.N.
      • Ballinger J.W.
      • Kim E.
      • Belchik S.M.
      • Lam K.S.
      • Youn B.
      • Nissen M.S.
      • Xun L.
      • Kang C.
      ), whereas for the structure of the oxygenase component (HsaA) of HsaAB, the enzymes involved in cholesterol catabolism in M. tuberculosis (
      • Dresen C.
      • Lin L.Y.
      • D'Angelo I.
      • Tocheva E.I.
      • Strynadka N.
      • Eltis L.D.
      ), His-368 and Ser-143 are at positions equivalent to His-396 and Ser-171 in C2. His-101 and Ser-171 were identified as potential catalytic residues in the active site of kijD3 (
      • Bruender N.A.
      • Thoden J.B.
      • Holden H.M.
      ,
      • Hu Y.
      • Al-Mestarihi A.
      • Grimes C.L.
      • Kahne D.
      • Bachmann B.O.
      ). These enzyme structures are shown in supplemental Fig. S3. These data suggest that His-396 and Ser-171 of C2 are conserved and that their functional roles may be prevalent in other two-component flavin-dependent monooxygenases.
      Our results (Table 1 and FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6) show that His-396 is important for the formation of C4a-hydroperoxy-FMN but is less likely to have a direct role in the H2O2 elimination process. Table 1 shows that the rate constants of C4a-hydroperoxy-FMN formation in the His-396 mutants are significantly lower than the C2 wild type. Lesser effects were found for the rate constant of the H2O2 elimination step. All mutants bind well to FMNH except for the H396R and H396K mutants in which the long side chains of Arg and Lys may have caused steric hindrance, which obstructed the flavin binding (supplemental Figs. S1 and S2). The reactions of reduced H396N and reduced H396V with oxygen showed that at all pH values employed, the reduced flavin bound to the mutants was primarily in the anionic form (deprotonated at flavin N1; Fig. 1) and that pKa value of the reduced flavin was lower than 6.0 because rate constants for the formation of C4a-hydroperoxy-FMN in both mutants were independent of pH (Fig. 5, similar to the C2 wild type (
      • Ruangchan N.
      • Tongsook C.
      • Sucharitakul J.
      • Chaiyen P.
      ). The difference was found for the reaction of reduced H396V at pH 6.0, in which the rate constant for C4a-hydroperoxy-FMN formation was lower than at other pH values (2.1 versus 4.4 × 103 m−1 s−1). In this case, at pH 6.0 the reduced FMN may exist as a neutral form (Fig. 5). According to Bruice (
      • Bruice T.C.
      ) and Venkataram and Bruice (
      • Venkataram U.V.
      • Bruice T.C.
      ), only the anionic form of the reduced FMN reacts rapidly with oxygen to form a radical pair of flavin semiquinone and superoxide that eventually forms C4a-hydroperoxy-FMN. We also speculate that the effect of His-396 mutation may be on the protonation status of a peroxide group of C4a-hydroperoxy-FMN because the reaction of H396N and H396V showed an interesting absorbance decrease of C4a-hydroperoxy-FMN at 390 nm with a rise in pH (Fig. 5), in contrast to the C2 wild-type kinetic traces, which did not show any intermediate absorption change at various pH values (
      • Ruangchan N.
      • Tongsook C.
      • Sucharitakul J.
      • Chaiyen P.
      ).
      The pH-dependent studies of reduced H396N and reduced H396V with oxygen (FIGURE 5, FIGURE 6) have indicated that His-396 is clearly not a general base involved with the H2O2 elimination process in C2. For both mutants, the rate constants of the H2O2 elimination step (step 2 in Fig. 1) increased upon a rise in pH and were correlated with a pKa of >9.7 for H396N and >9.6 for H396V. These pH dependences are similar to the pattern found for the C2 wild-type enzyme (pKa >9.4). Therefore, His-396 clearly does not act as a general base to assist in the process of H2O2 elimination. In fact, having no base to catalyze the H2O2 elimination, as in this case, is functionally advantageous for the two-component monooxygenase because it helps prolong the lifetime of the C4a-hydroperoxyflavin. This prolonged lifetime helps to reduce the production of wasteful H2O2 when a substrate is absent and ensure efficiency in the hydroxylation in the presence of a substrate. This statement is confirmed by results of DHPA analysis. Stabilization of C4a-hydroperoxy-FMN is crucial for DHPA formation because the mutants or conditions with low stability of the intermediate do not promote efficient hydroxylation (Table 2).
      Results in FIGURE 7, FIGURE 8, FIGURE 9 and Table 1 clearly show that Ser-171 in C2 is a major residue that stabilizes the C4a-hydroperoxy-FMN. The stabilization energy of C4a-hydroperoxy-FMN is most likely contributed by the H-bond interaction at the flavin N5 via Ser-171. A single mutation of Ser-171 to Ala showed a 1400-fold increase for the rate constant of H2O2 elimination, whereas an 8-fold increase was found for the S171T mutant, in which a hydroxyl group side chain was still present. The results from the double mutant S171A/H396V also agree with this conclusion because the double mutant failed to form the C4a-hydroperoxy-FMN intermediate. For the double mutant S171A/H396V, a rate constant of C4a-hydroperoxy-FMN formation was predicted to be ∼300-fold lower based on the H396V mutation, and the intermediate decay was predicted to be ∼1400-fold higher based on the S171A mutation. Such effects may cause the rate constant of the intermediate decay to supersede the intermediate formation, preventing kinetic detection of the intermediate. Kinetic traces of Fig. 9 did not show any kinetic detection of C4a-hydroperoxy-FMN, confirming the roles of His-396 and Ser-171 proposed, and suggest that a hydroxyl group of Ser-171 is a crucial feature stabilizing C4a-hydroperoxy-FMN.
      The formation of a hydrogen-bond between the Oγ of Ser-171 and the flavin N5-H is likely a key feature that stabilizes the C4a-hydroperoxy-FMN intermediate in C2 (Fig. 10). Based on the three-dimensional structure of C2, the Oγ atom of Ser-171 is located ∼2.9 Å away from flavin N5. This distance allows the formation of a hydrogen bond between Ser-171 Oγ and flavin N5 (Fig. 2). Recently, studies of pyranose 2-oxidase (
      • Sucharitakul J.
      • Wongnate T.
      • Chaiyen P.
      ) using transient kinetics and kinetic isotope effects have shown that the bond breakage of the flavin N5-H is the rate-limiting step controlling the overall process of H2O2 elimination from C4a-hydroperoxyflavin. It was proposed that the mechanism of H2O2 elimination involved an intramolecular proton bridge transfer to generate a H2O2 leaving group (Fig. 10) (
      • Sucharitakul J.
      • Wongnate T.
      • Chaiyen P.
      ). For pyranose 2-oxidase, mutations of Thr-169, a residue that is located ∼3 Å away from the flavin N5 similar to Ser-171 in C2, abolished the formation of C4a-hydroperoxyflavin (
      • Pitsawong W.
      • Sucharitakul J.
      • Prongjit M.
      • Tan T.C.
      • Spadiut O.
      • Haltrich D.
      • Divne C.
      • Chaiyen P.
      ). Based on this view, we predict that the role of Ser-171 in C2 is for the hydrogen bonding interaction with flavin N5-H to impede the deprotonation at the flavin N5-H. At low pH (Fig. 10), elimination of H2O2 may occur via intramolecular proton bridge transfer, similar to the model described for pyranose 2-oxidase reaction (
      • Sucharitakul J.
      • Wongnate T.
      • Chaiyen P.
      ). For elimination of H2O2 at high pH, a hydroxide ion acts as a specific base to catalyze the process, causing an increase in rate constants as pH increases (Fig. 10).
      Figure thumbnail gr10
      FIGURE 10The model proposed for the stabilization of the C4a-hydroperoxy-FMN intermediate and mechanisms of H2O2 elimination at low and high pH levels. The picture shows a hydrogen bond interaction between the flavin N5 proton and the Ser-171 side chain that impedes the flavin N5-H bond breakage, which in turn obstructs the H2O2 elimination process. At low pH, the H2O2 elimination occurs through an intramolecular hydrogen atom transfer as proposed for pyranose 2-oxidase reaction (
      • Sucharitakul J.
      • Wongnate T.
      • Chaiyen P.
      ). At higher pH, the H2O2 elimination is faster because the concentration of a hydroxide ion that deprotonates the flavin N5 proton increases, and thus, the rate constant of the process increases.
      Studies of pH effects on the reaction of S171A (Fig. 8) also support the role of Ser-171 proposed in Fig. 10 because a pKa-dependent pattern associated with the elimination of H2O2 in the S171A mutant is significantly different from those found for the C2 wild-type enzyme and His-396 mutants (FIGURE 6, FIGURE 8). However, the pKa values obtained from FIGURE 6, FIGURE 8 do not reflect the real pKa of the ionizable group (Fig. 10) because the upper limits of the plots could not be reached because of experimental limitations. For the wild-type and His-396 mutant enzymes, conditions at pH greater than 10.0 denatured the enzyme. For the S171A mutant, at pH 8.0 and above, little of C4a-hydroperoxy-FMN was formed, causing a false end point. Nevertheless, the data suggest that by removing a hydrogen bonding of Ser-171 and flavin N5, the deprotonation of flavin N5-H and thus H2O2 elimination is more favorable at all pH values compared with the wild-type enzyme.
      Assignment of an ionizable group associated with the pH-dependent plots in FIGURE 6, FIGURE 8 is not definitive based on the current data because the upper limit value could not be reached. One possible interpretation is that, at higher pH, the concentration of hydroxide increases, promoting deprotonation of flavin N5-H in the transition state as depicted in Fig. 10. Thus, the pH-dependent plots in FIGURE 6, FIGURE 8 do not reflect a pKa of an ionizable group but merely a specific base catalysis. Results in Fig. 8 support this model because the lack of H-bonding at flavin N5 in the S171A mutant would make deprotonation of the flavin N5-H by a specific base easier than in the C2 wild type. The hydrogen bonding interaction between the flavin N5-H and a hydrogen bond acceptor side chain (Fig. 10) may be an important feature for the stabilization of a long-lived C4a-hydroperoxyflavin in other flavin-dependent monooxygenases. The structure of HpaB from T. thermophilus HB8 (2YYI), a two-component enzyme that catalyzes similar hydroxylation as C2 but with sequence identity of only 11.6%, shows that the Oγ of Thr-185, a homologous residue of Ser-171 in C2, is 2.8 angstrom from the flavin N5 (
      • Kim S.H.
      • Hisano T.
      • Takeda K.
      • Iwasaki W.
      • Ebihara A.
      • Miki K.
      ) supplemental Fig. S3). Although the reaction kinetics of HpaB from T. thermophilus HB8 has not been reported, kinetics of HpaA, the oxygenase component of HPAH from P. aeruginosa, which is similar to HpaB from T. thermophilus (28.5% identity) (
      • Chakraborty S.
      • Ortiz-Maldonado M.
      • Entsch B.
      • Ballou D.P.
      ), is known in detail. The results have shown that C4a-hydroperoxyflavin in this enzyme is quite stable, with a rate constant for H2O2 elimination of 0.005 s−1. It is possible that Thr-185 of HpaB is responsible for C4a-hydroperoxyflavin stabilization. The previous study of HPAH from P. aeruginosa also mentioned the possibility that the hydrogen bond to flavin N5 is responsible for stabilization of C4a-hydroperoxyflavin (
      • Chakraborty S.
      • Ortiz-Maldonado M.
      • Entsch B.
      • Ballou D.P.
      ). Recently, studies of a flavin-containing monooxygenase have shown that the mutation that alters the H-bond interactions between the flavin N5 and NADP+ also resulted in an enzyme that does not form C4a-hydroperoxyflavin (
      • Orru R.
      • Pazmiño D.E.
      • Fraaije M.W.
      • Mattevi A.
      ). For cyclohexanone monooxygenase in which C4a-hydroperoxyflavin is stabilized (
      • Sheng D.
      • Ballou D.P.
      • Massey V.
      ), the enzyme structure in a complex with NADP+ has shown close interactions between NADP+ and flavin N5 (supplemental Fig. S3) (
      • Mirza I.A.
      • Yachnin B.J.
      • Wang S.
      • Grosse S.
      • Bergeron H.
      • Imura A.
      • Iwaki H.
      • Hasegawa Y.
      • Lau P.C.
      • Berghuis A.M.
      ). A summary for kinetic constants associated with the H2O2 elimination in various enzymes is shown in supplemental Table S3.
      In conclusion, our findings here present experimental evidence showing that the conserved His-396 and Ser-171 in the active site of the oxygenase component of HPAH from A. baumannii (C2) are important for the formation and stabilization of C4a-hydroperoxy-FMN. His-396 facilitates the formation of C4a-hydroperoxy-FMN and is not involved in the H2O2 elimination process. The hydrogen bonding between Ser-171 and the flavin N5 is a key interaction for prolonging the life time of C4a-hydroperoxy-FMN. The presence of residues homologous to these two residues in the active sites of other two-component monooxygenases implies that their functional roles may be prevalent in other monooxygenases.

      Acknowledgments

      We thank Barrie Entsch (University of New England, New South Wales, Australia) for critical reading of the manuscript.

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