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ABBArchives of Biochemistry and Biophysics 442 (2005) 102–116

Mechanism of flavin transfer and oxygen activationby the two-component flavoenzyme styrene monooxygenase

Auric Kantz, Franklin Chin, Nagamani Nallamothu,Tim Nguyen, George T. Gassner *

Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132-4163, USA

Received 21 June 2005, and in revised form 21 July 2005Available online 18 August 2005

Abstract

Styrene monooxygenase (SMO) from Pseudomonas putida S12 is a two-component flavoenzyme composed of the NADH-specificflavin reductase, SMOB, and FAD-specific styrene epoxidase, SMOA. Here, we report the cloning, and expression of native andhistidine-tagged versions of SMOA and SMOB and studies of the flavin transfer and styrene oxygenation reactions. In the reductivehalf-reaction, SMOB catalyzes the two-electron reduction of FAD with a turnover number of 3200 s�1. Single turnover studies ofthe reaction of reduced SMOA with substrates indicate the formation of a stable oxygen intermediate with the absorbance charac-teristics of a flavin hydroperoxide. Based on the results of numerical simulations of the steady-state mechanism of SMO, we find thatthe observed coupling of NADH and styrene oxidation can be best explained by a model, which includes both the direct transfer andpassive diffusion of reduced FAD from SMOB to SMOA.� 2005 Elsevier Inc. All rights reserved.

Keywords: Electron transfer; Flavin monooxygenase; Epoxidation; Enzyme intermediate; Redox; Pre-steady-state kinetics; Transient kinetics;Styrene; Reductase; Singlet oxygen

Pyridine nucleotide-dependent oxygenases haveevolved in prokaryotes as a diverse group of enzymesexisting in both soluble and membrane-bound forms.Structurally, they range from self-contained single-com-ponent enzymes to complex systems including separatereductase, electron-transfer, regulatory, and oxygenaseactivities. These enzymes engage heme, non-heme iron,copper, or flavin-dependent active sites in their substrateoxygenation reactions [1–5].

Flavin-dependent monooxygenases catalyze reactionsranging from the genesis of bioluminescence to the cat-abolic oxidation of hydrocarbons [6,7]. Single-compo-nent flavoenzymes house both flavin reduction andsubstrate oxygenation activities in the same contiguouspeptide and have been shown to proceed through flavin

0003-9861/$ - see front matter � 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.abb.2005.07.020

* Corresponding author. Fax: +1 415 338 2384.E-mail address: [email protected] (G.T. Gassner).

C-4a hydroperoxide and hydroxide intermediates in thesubstrate oxygenation reaction [7]. Similar intermediateshave been reported and proposed for the closely relatedtwo-component systems [8–12].

Two distinct classes of two-component flavoenzymeshave been identified. The first of these, exemplified by 4-hydroxyphenylacetate-3-monooxygenase from Pseudo-monas putida, is composed of a separate oxygenaseand regulatory protein required for the efficient couplingof pyridine nucleotide oxidation and substrate oxygena-tion activities [9,13]. More commonly encountered two-component flavoenzymes are composed of distinct flavinreductase and monooxygenase activities segregated onseparate peptide units [6,10–12,14–16].

The spatial resolution of flavin reduction and sub-strate oxygenation activities in the two-component fla-vin monooxygenases requires either a direct or adiffusive transfer of reduced flavin from the reductase

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A. Kantz et al. / Archives of Biochemistry and Biophysics 442 (2005) 102–116 103

to the monooxygenase. Reductase components of theseenzymes have been distinguished based on differencesin their mechanisms of interaction with flavin substrates[17]. Analysis of the steady-state mechanisms of bacte-rial luciferase and alkanesulfonate monooxygenase sug-gests that these systems operate through a mechanism ofdirect transfer of reduced flavin [16,18,19].

Two-component styrene monooxygenases (SMOs)1

from Pseudomonas are similarly composed of highly con-served flavin reductase and epoxidase components. It hasbeen suggested that in these enzymes and closely relatedsystems including 4-hydroxyphenylacetate-3-monooxy-genase isolated from Escherichia coli W flavin transferfrom the reductase to oxygenase component is purely dif-fusive [12,15,20]. In support of this mechanism it hasbeen shown that the monooxygenase component ofSMO is capable of substrate epoxidation in the absenceof reductase when provided with an alternate source ofreduced FAD [21]. However, rapid reactions of reducedFAD with oxygen and oxidized FAD are likely to makethe diffusive mode flavin transfer quite inefficient [7].

Substrate epoxidation is a common reaction of boththe heme and non-heme iron-containing enzymes [22–25], but unusual for flavoenzyme monooxygenases.Studies of the oxygen reaction of SMO may provide in-sight into flavin-based epoxidation mechanism of thisenzyme and other flavoenzyme epoxidases such as squa-lene and zeaxanthin monooxygenases [26,27].

Here, we report the cloning expression of native andN-terminally histidine-tagged versions of the styrenemonooxygenase reductase and epoxidase componentsof P. putida S12. Kinetic data recording the time-depen-dent evolution and spectral characteristics of oxygenintermediates are presented. To further explore the modeof flavin transfer in this system, the observed efficiency ofcoupling NADH oxidation to styrene consumption overa range of enzyme component ratios and FAD concen-trations is compared with the results of simulations of di-rect and diffusive flavin-transfer mechanisms.

Materials and methods

Cell growth, protein, and DNA purification from native P.

putida S12

Pseudomonas putida S12 was purchased from theATCC [28]. This strain of cells grows poorly in culturewith styrene as the sole source of carbon and energyand expression from the styrene operon is subdued bycatabolite repression by alternate carbon sources foundin enriched media [29]. The styrene operon of P. putidaS12 is not catabolite-repressed by phenyl acetic acid [30],

1 Abbreviations used: SMO, styrene monooxygenase; PMSF, phen-ylmethylsulfonyl fluoride; EDTA, ethylenediaminetetraacetic acid.

but the cells grow poorly in liquid culture when limitedto this substrate.

To circumvent these limitations, cells were initiallygrown in Trypticase-Soy Broth supplemented with0.1% phenyl acetic acid in a 10 L New Brunswick Bio-Flow 2000 Fermenter. Approximately 130 g of cell pastewas recovered by centrifugation following a 16-h growthperiod at 33 �C. The cell paste was then resuspended inminimal media containing phenylacetic acid as the onlycarbon source. After a half hour equilibration time, thestyrene operon was induced by the addition of styrene tothe cell culture to a final concentration of 1 mM. Fol-lowing a 1-h induction period, approximately 130–150 g of cell paste was recovered.

Cells were resuspended in 20 mM phosphate buffer(pH 7) containing 1 mM of each phenylmethylsulfonylfluoride (PMSF) and ethylenediaminetetraacetic acid(EDTA) and disrupted by sonication for a total timeof 6 min, while maintaining the solution temperature be-low 10 �C. Particulate cell debris was removed from thesoluble protein fraction by centrifugation for 45 min at18,000 rpm in an SS34 rotor at 4 �C. Ultimately nativestyrene monooxygenase components were partially puri-fied by anion exchange, dye-ligand, and reverse-phasechromatography.

Cloning design of expression vectors and sequencing

DNA was isolated from P. putida S12 after sub-cul-turing on agar plates supplemented with 0.1% phenylacetic acid and 1 mM indole. Indigo producing cellswere recovered by centrifugation and subjected to alka-line lysis and genomic DNA extraction by using a Qia-gen Genomic Tip protocol. Primers were designedbased on the reported sequences of styA and styB fromPseudomonas fluorescens [31] and purchased from Oper-on. The forward and reverse primers used for the ampli-fication of styA were 5 0-CCATATGAAAAAGCGTATCGGTATT-GTTGGTG-3 0 and 5 0-CCTTAAGTCAGGCCGCGATAGTGGGTGC-3 0, respectively. Forwardand reverse primers for the amplification of styB were5 0-CCATATGACGTTAAAAAAAGATATGGCGGTGG-3 0 and 5 0-CCTTAAGTTAATTCAGCGGCAACGGGTTAC-3 0. In each case, the primers were de-signed to introduce an NdeI site at the 5 0-end of theamplified styA and styB genes. The styA and styB geneswere amplified from genomic DNA by 30 cycles of PCRwith Pfu-Turbo polymerase. PCR products were recov-ered after gel purification by using Q-Biogene silica gelspin-columns. The purified PCR products were 5 0-phos-phorylated by treatment with T4 polynucleotide kinaseand inserted by blunt-end ligation with T4 ligase intothe EcoRV site of pZErO-2 vector purchased from Invit-rogen. E. coli TOP-10 cells were transformed and posi-tive clones were selected. pZErO-2 vectors containingstyA and styB were recovered by the Qiagen mini prep

104 A. Kantz et al. / Archives of Biochemistry and Biophysics 442 (2005) 102–116

plasmid purification protocol. The styA and styB geneswere then ligated into the NdeI and EcoRI restrictionsites in the multiple cloning site of pET-28 and pET-29expression vectors from Novagen. This work yieldedvectors pET-29SMOA and pET-29SMOB designed forthe synthesis of native SMOA and SMOB and vectorspET-28NSMOA and pET-28NSMOB for the synthesisof thrombin cleavable, N-terminally histidine-taggedSMOA and SMOB. Sequencing primers were designedand used to verify the styA and styB gene sequences inthe recombinant expression vectors. It was determinedthat several mutations were introduced due to differenc-es in the P. fluorescens and S12 DNA sequences. How-ever, the encoded amino acid sequences of SMOA andSMOB were unaffected due to degeneracy of the geneticcode translation. The sequence of styA from P. putidaS12 was previously reported (GenBank Accession No.Y13349). Our results confirm this sequence. The SMOBsequence was determined to be identical to that of styBisolated from Pseudomonas sp. VLB [32].

Expression and purification of recombinant proteins

Native and N-terminally histidine-tagged forms ofSMOA were expressed in E. coli BL21(DE3) cells. Typ-ically, a yield of 30 mg of protein per liter was recoveredafter a 1-h induction period in growth medium supple-mented with 30 lg mL�1 of ampicillin and 1 mM IPTGat 37 �C. A large quantity of indigo dye accumulateswhen these proteins are expressed in LB medium andfor this reason SMOA was typically expressed in M-9medium. Native and N-terminally histidine-taggedSMOB was expressed from E. coli BL21 (DE3) underthe same conditions. SDS–PAGE assay of soluble andinsoluble cell fractions recovered after expression indi-cated that whereas SMOA and N-SMOA are over-ex-pressed as purely soluble protein, the over-expressedSMOB and N-SMOB primarily form inclusion bodies.Only about 1% of the SMOB expressed remains in thesoluble fraction, and we were unsuccessful in our effortsto increase the fraction of protein expressed in solubleform by inducing the cells to express protein at lowertemperatures. It was possible to recover soluble, activeSMOB from inclusion bodies by denaturation in 8 Murea containing 5 mM DTT as previously reported[15]. N-terminally histidine-tagged SMOA and SMOBwere recovered from 4–10 L cell growths in M-9 medi-um. Cell pellets were resuspended and sonicated in Ni–NTA equilibration buffer composed of 50 mM sodiumphosphate, 10 mM imidazole, 300 mM sodium chloride,1 mM PMSF, and 1 mM EDTA. Immediately after cen-trifugation, soluble extracts containing N-His-SMOAand N-His-SMOB were pumped onto a 1.5 · 5 cmOmnifit column containing freshly charged Sigma His-Select resin at a flow rate of 3 mL min�1. The columnwas then washed with one column volume of equilibra-

tion buffer followed by a linear 50 mL gradient of imid-azole from 10 to 250 mM. Protein fractions were pooledbased on UV absorbance and SDS–PAGE assay andstored at �80 �C after addition of dithiothreitol to a fi-nal concentration of 1 mM and glycerol to 50%.

Protein concentration measurements and thiol titration

Molecular weights and extinction coefficients forSMO components were estimated by using the web-based application Prot Param [33]. Protein concentra-tions were calculated by using these values and by thePierce BCA assay with BSA as a protein standard.Accessible thiols were titrated with 5 0,5 0-dithionitroben-zoic acid with dithiothreitol as a thiol standard.

Steady-state reaction mechanism of SMOB

The steady-state reaction of native SMOB with an ar-ray of NADH and FAD concentrations was monitoredby stopped-flow absorbance spectroscopy. In these stud-ies, apo enzyme at a concentration of 10 nM after mix-ing was reacted with defined substrate mixtures inair-saturated 20 mM phosphate buffer, pH 7, containing5% glycerol and 1 mM dithiothreitol. A stopped-flowspectrophotometer equipped with an Ocean OpticsUSB-2000 diode array spectrophotometer was used formixing and data collection. Absorbance changes corre-sponding to NADH reduction were monitored at340 nm, while the FAD oxidation state was monitoredat 450 nm. Initial rates were calculated from linear fitsthrough the first 5–10 s of absorbance data recordingthe oxidation of NADH to NAD+. No significantreduction of FAD was detected over this time perioddue to the rapid reoxidation of FADH by molecularoxygen. Three to five replicate reactions were analyzedfor each experimental condition. The reaction rate datacorresponding to each experimental NADH and FADconcentrations were entered into a single data arrayand fit globally along each substrate concentration axisto the function describing a sequential bimolecular mod-el (Eq. (1)), in which the apparent Vmax and KM valuesare given by Eqs. (2) and (3).

v ¼ Vappmax½NADH�

KappM þ ½NADH�; ð1Þ

V appmax ¼V max½FAD�

KFADM þ ½FAD�; ð2Þ

KappM ¼KFADM K

NADHS þ KNADHM

KFADM þ ½FAD�. ð3Þ

Data points analyzed in these fits were weighted basedon standard deviation from the mean experimental val-ues by using the program GraphPad Prism 4 (GraphPadSoftware). This global-fitting approach provides clear


advantages over alternate methods that rely on datamanipulation to generate linear plots, sequential, inde-pendent fitting of data sets, and construction of second-ary kinetic plots [34].

Pre-steady-state kinetic analysis of SMOA

Equal concentrations of N-terminally histidine-tagged SMOA and oxidized FAD were combined andmade anaerobic in a tonometer equipped with a titrationport and 1 cm pathlength quartz cuvette by repeatedlyevacuating and back filling with purified nitrogen gason a vacuum Schlink line. The SMOA–FAD complexwas reduced by incremental addition of dithionite froma gas-tight Hamilton syringe attached to the tonometerthrough a ground glass joint. Stock dithionite solutionswere prepared by addition of solid dithionite to an anox-ic buffer solution prepared by exhaustive bubbling withnitrogen and confirmed by measuring the absorbanceat 314 nm using an extinction coefficient of8000 M�1 cm�1 [35]. The extent of flavin reduction wasrecorded by monitoring the absorbance changes at 450and 314 nm. Reductive titrations were halted just priorto complete FAD reduction or just after the first detect-able increase in absorbance 314 nm was observed corre-sponding to the accumulation of 5–10 lM excessdithionite. Reduced enzyme solutions were transferredto a double-mixing stopped-flow spectrophotometer de-signed and constructed at San Francisco State Universi-ty. In test reactions we measured a 3.2 ms dead time forthis instrument, which is adequate for the studies de-scribed in this paper. The plumbing throughout theinstrument is PEAK plastic excepting the drive syringeswhich are 2.5 mL Hamilton syringes. The heart ofstopped flow is a mixer and 20 lL flow cell equipped withfiber optic cables that allow absorbance measurements tobe made across a 1 or 0.35 cm optical path length. Theplumbing and flow cell are completely contained in athermostated water bath. During anaerobic studies, thewater bath was continuously sparged with industrial-grade nitrogen. Under these conditions, flavin solutionscontained in the plumbing and flow cell of the instrumentremained fully reduced over the course of several hours.

This instrument was used in single-mixing mode torapidly mix equal volumes reduced SMOA with aerobicbuffer solutions containing oxygen and styrene or ben-zene. The flow cell was illuminated with a 75 W Xenonlamp and absorbance changes were monitored by aUSB2000 diode array spectrophotometer interfacedwith the stopped-flow spectrophotometer through solar-ization-resistant fused silica optical fibers purchasedfrom Polymicro Technologies. Each processed absor-bance spectrum represents the average of seven rawspectra each recorded at a frequency of 333 s�1. Noisederiving from small fluctuations in xenon lamp intensitywas diminished by reference monitoring. The total effec-

tive 21 ms exposure time was short enough to resolve thefastest reactions detected in our studies and long enoughto provide a good signal-to-noise ratio.

Kinetic data were fit to functions consisting of sumsof 3–4 exponentials (Eq. (4)) by using the program Kale-idaGraph (Synergy Software).

Aðk;tÞ ¼Xin¼1

an e�knt þ const. ð4Þ

Observed rate constants obtained in this way were usedto estimate the time-dependent changes in concentra-tions in the sequential transformation of reactants tointermediate species and products by using the KINSIMprogram [36,37]. The resulting concentration tables werethen referenced to find the best-fitting extinction coeffi-cients corresponding to each intermediate spectrum byusing the table function built into the KaleidaGraphprogram.

Efficiency of coupling NADH and styrene oxidation

reactions of SMO

The steady-state kinetic reaction of the two-compo-nent styrene monooxygenase system was recorded byusing a Ocean Optics DT-1000 deuterium lamp andUSB2000 diode array spectrophotometer interfacedwith the stopped-flow instrument described above. Thestopped-flow instrument allowed precise 1:1 mixingand measurement of initial rates and the control of tem-perature, styrene, and oxygen concentration.

Steady-state kinetics were monitored simultaneouslyat 450 nm where only oxidized FAD hassignificant absorbance ðEM450 ¼ 11; 300 M�1 cm�1Þ, at340 nm where both FAD ðEM340 ¼ 4680 M

�1 cm�1Þ andNADH ðEM340 ¼ 6220 M

�1 cm�1Þ absorb significantly,and at 245 nm where FAD ðEM245 ¼ 18; 434 M

�1 cm�1Þ,NADH ðEM245 ¼ 10; 300 M

�1 cm�1Þ, NAD+ ðEM245 ¼12; 576 M�1 cm�1Þ, and styrene ðEM245 ¼ 8880 M�1 cm�1Þall absorb. Under conditions of aerobic steady-state turn-over, the absorbance at 450 nm remains constant at a val-ue corresponding to the initial concentration of oxidizedFAD included in the assay. For this reason, it was possibleto calculate reaction rate and coupling efficiency by con-sidering absorbance changes corresponding only to thedepletion of styrene and the transformation of NADHtoNAD+ at 245 and 340 nm.Data collected at higher sty-rene concentrations were recorded along the short(0.35 cm) path length of the stopped-flow flow cell to al-low the full dynamic range ofUV-absorbing reactant con-centrations to be studied. The rate of NADH oxidationwas calculated by dividing the time-dependent absor-bance change at 340 nm by the extinction coefficient forNADH at this wavelength. The rate of styrene oxidationwas calculated from the time-dependent absorbancechanges at 245 and 340 nmwith Eq. (5). The coupling effi-


ciency was determined by calculating the ratio of theNADH and styrene oxidation rates.

D½Styrene�Dt

¼eNADH245 � eNAD245� �eStyrene245 e

NADH340 b

� DA340Dt

� 1eStyrene245 b

� DA245Dt

. ð5Þ

Plots of styrene and NADH oxidation rate data wereanalyzed by non-linear regression analysis and bynumerical simulation according to the models describedin the text. The relatively small molar absorbtivitychange associated with styrene oxidation and the techni-cal difficulty of accurately preparing low-concentrationstyrene solutions set the limit of detection by this meth-od at 5–10 lM.

Oxidation–reduction potential measurements

The redox potentials of free- and enzyme-bound FADwere calculated from anaerobic titration with sodiumdithionite as the reductant. These studies were conductedat 25 �C in solutions buffered at pH 7 with 20 mMMOP-SO. Reactions typically included 20–50 lM concentra-tion FAD and one of a series of solution potentialindicators [38]. These studies were performed in a spe-cially designed quartz cuvette and made anaerobic byalternately drawing a vacuum and back flushing withwet, anaerobic nitrogen. Absorbance changes wererecorded during the titration at wavelengths correspond-ing to absorbance peaks of the oxidized indicator andFAD. The absorbance changes recorded at each pointin the titration were transformed into concentrationsby using Eqs. (6) and (7) in which the D represents thedifference between the oxidized and reduced value ofeach parameter. Each calculated concentration was cor-rected for dilution by multiplying by the ratio of the finalvolume after each addition of dithionite divided by theinitial volume prior to the addition of dithionite.

½FADred� ¼DAk2De

Dyek1

� DAk1DeDyek2

DeDyek1 DeFADk2

� DeFADk1 DeDyek2

; ð6Þ

½Dyered� ¼DAk1De

FADk2

� DAk2DeFADk1DeDyek1 De

FADk2

� DeFADk1 DeDyek2

. ð7Þ

The solution potential was calculated at each point inthe titration by entering the calculated dye concentra-tions into the Nernst equation.

Results

Protein expression and purification

More than 95% of the total native and N-terminallytagged versions of SMOB are expressed in the form of

insoluble inclusion bodies. Native SMOB we recoveredby resolubilization from inclusion bodies had very simi-lar catalytic activity compared with the soluble fractionof N-terminally tagged SMOB recovered by nickel–NTA affinity chromatography.

Enzymes were stored in 50% glycerol at �80 �C in aconcentration of 1–5 lM. Native and N-terminallytagged SMOB tended to aggregate and lose activitywhen concentrated beyond this level centrifugally orby reverse osmosis. Dilute solutions of the native andengineered versions of SMOB were stabilized in assaybuffers containing 5% glycerol and 1 mM dithiothreitolfor use in initial rate measurements.

In the presence of phosphate buffer, SMOA precipi-tates and for this reason experiments including SMOAwere buffered with MOPSO. Native and N-terminallyhistidine-tagged versions of SMOA were found to besoluble and amenable to concentration. The engineeredversion of SMOA behaves very similarly to the nativeprotein in kinetic assays (Table 1), and since it was sig-nificantly easier to isolate the histidine-tagged protein,we elected to use it rather than the native protein inthe studies described in this paper. A picture of a gelshowing purified native and histidine-tagged proteins isshown in Fig. 1.

Steady-state mechanism of SMOB

The pH profile of SMOB reacting with NADH andFAD suggests an ideal operating pH between 6 and 7.By fitting the pH profile between pH 5 and 8 it was pos-sible to calculate the value of macroscopic pKa values of4.4 ± 0.2, 7.6 ± 0.1, and 8.2 ± 0.2 associated with theVmax and 6.1 ± 1.2, 7.9 ± 3.3, and 8.9 ± 1.9 associatedwith KM (Fig. 2). In these studies, 20 nM SMOB wascombined with 120 lM FAD in 5 mM MOPSO bufferat pH 7 containing 5% glycerol and 1 mM DTT. The en-zyme–FAD complex was then rapidly mixed in thestopped flow with each of a series of solutions containinga 100 mM pH defining buffers including citrate, MES,POPSO, CHES, CAPS, and various concentrations ofNADH. The exact pH values after mixing were measuredexperimentally. Since these data were recorded at onlyone FAD concentration, the pKa values correspond onlyto apparent values of Vmax and KM and cannot be as-signed to individual steady-state parameters.

Product inhibition studies were used to characterizethe sequence of substrate binding and product releasein the reaction of SMOB with NADH and FAD(Fig. 3A). These results suggest that NAD+ acts as acompetitive inhibitor of NADH and mixed inhibitor ofFAD in its interaction with SMOB. This inhibition pat-tern is consistent with the sequential bimolecular reac-tion mechanism in which NADH serves as the leadingsubstrate as previously reported for the reaction ofFMN with SMOB from the VLB strain [15].

Table 1Comparison of native and N-terminally histidine-tagged SMOA

SMOA (Vmax/KM)app (s�1)a (Vmax/KM)app (s

�1)b [NADH]/[Styrene]c [NADH]/[Styrene]d

Native 0.07 ± 0.03 0.02 ± 0.01 0.9 ± 0.1 6.7 ± 2.7N-His 0.05 ± 0.01 0.05 ± 0.03 1.1 ± 0.1 4.2 ± 0.9

Steady-state reactions included 10 nM native SMOB 0.5 lM native or N-terminally histidine-tagged SMOA and 50 lMNADH. aKinetic parametersfrom reactions which included 10 lM FAD and styrene as the varied substrate. bKinetic parameters from reactions which included 20 lM styreneand FAD as the varied substrate. Coupling of NADH consumption to styrene oxidation in reactions including 10 lM FAD and 100 lM cstyrene or20 lM styrene and 100 lM dFAD.

Fig. 1. SDS–PAGE 4–20% acrylamide gradient gel of purified SMOcomponents. The contents of each lane are as follows: Lane 1,molecular weight markers; lane 2, native SMOB after concentrationand purification through a BioRad Uno-Q anion exchange column;lane 3, N-terminally histidine-tagged SMOB after resolution by Ni–NTA chromatography; lane 4, molecular weight markers; lane 5,native SMOA after partial purification by anion exchange chroma-tography; and lane 6, N-terminally histidine-tagged SMOA afterpurification by Ni–NTA chromatography.

Fig. 2. pH profile of the reaction of native SMOB with NADH andFAD. Apparent values of Vmax (s) and KM (h) determined at 25 �Cand pH values of 5.2, 5.6, 6.2, 6.7, 7.2, 7.6, 7.9, 8.3, and 9.2 were fit asdescribed in the text. Solid lines represent the best fits through thesedata. Dashed line represents the Vmax/KM ratio calculated as afunction of pH.


Global fits through initial rate data from reactions ofSMOB with various concentrations of FAD and NADHare plotted in Fig. 3B. The best-fitting steady-state kinet-ic parameters and their experimental uncertainties were:kcat = 3240 ± 650 lM s

�1, KM(NADH) = 101 ± 53 lM,Ki (NADH) = 114 ± 41 lM, KM(FAD) = 86 ± 29 lM.

Steady-state analysis of SMOA

A plot of reaction velocity of SMO as a function ofstyrene concentration is presented in Fig. 4A. SMOAconcentration was 50-fold greater than SMOB and theflavin concentration was maintained at a low level soas to maximize the efficiency of coupling NADH oxida-tion to styrene consumption. Under these conditions,NADH/styrene ratio was found to decrease fromapproximately 1.7 in the low range of styrene concentra-tion to 1.0 in the presence of saturating styrene. Best-fit-ting parameters gave an apparent KM for styrene of4.6 ± 0.7 lM and Vmax of 200 ± 5 nM s

�1.Benzene was determined to be a competitive inhibitor

of styrene. Fig. 4B shows a fit of initial velocity datafrom the reaction of SMOA with styrene over a rangeof benzene concentrations. These data were fit accordingto the equation for simple substrate inhibition to calcu-late an estimate of the equilibrium dissociation constantfor benzene. In this way, a dissociation constant of173 ± 10 lM was determined.

Pre-steady-state investigation of SMOA

Reaction of reduced SMOA with oxygen was investi-gated by single-mixing stopped-flow experiments. In thesestudies, solutions containing equimolar concentrations ofSMOA and FAD were first titrated in an anaerobic cuv-ette and then transferred to the stopped flow where theywere rapidly mixed with aerobic buffer solutions. Titra-tions performed in the presence of a small amount ofresidual oxygen resulted in the formation of a stable inter-mediate spectrumwith an absorbancemaximum centeredbetween 370 and 380 nm, asmight be expected for a flavinhydroperoxide [39–41]. Under anaerobic conditions, thisintermediate remained stable and showed no evidence ofreduction by dithionite. Under aerobic conditions, theintermediate was observed to decompose. The rate of

Fig. 3. Steady-state mechanism of SMOB reacting with NADHand FAD. (A) Product inhibition studies with NAD included as inhibitor of substratesNADH (h) and FAD (s). (B) Global fit of the bi-sequential model through initial velocity data from the reaction of 10 nM SMOB with NADHconcentrations ranging from 20 to 350 lM and FAD concentrations ranging from 1 to 100 lM in 20 mM phosphate buffer at pH 7 and 25 �C.

Fig. 4. Steady-state reaction of SMO with styrene and inhibition by benzene. Reaction of 0.5 lM N-terminally histidine-tagged SMOA and 10 nMnative SMOB with 10 lM FAD, 50 lM NADH, and 270 lM oxygen, and styrene in 20 mM aerobic MOPSO buffer pH 7 at 25 �C. (A) Michaelis–Menten fit through initial rate data from reactions, which included styrene at concentrations ranging from 5 to 100 lM. (B) Competitive inhibition fitthrough data from reactions, which included 15.6 lM styrene and benzene concentrations ranging from 20 to 500 lM.


decomposition was accelerated by the addition of styreneand inhibited by the addition of benzene.

Kinetic data recorded at 314, 390, and 450 nm areshown in Figs. 5A–C. These data illustrate the differentreaction kinetics, which occur in the oxidative reactionof SMOA with air in the presence or absence of styreneor benzene. Solid lines passing through the data in eachplot represent the best three or four exponential fitsthrough these data and the best-fitting rate constantscorresponding to each fit are listed in Table 2. Extinc-tion coefficients corresponding to the initial, intermedi-ate, and final spectra involved in each reaction aregiven in Figs. 5D–F.

Measurement of the Redox potential of FAD associatedwith SMOA

Titration of N-terminally histidine-tagged SMOA inthe presence of FAD and anthraquinone-2-sulfonate

resulted in the sequential reduction first of enzyme-bound FAD and then the indicator. It was clear fromthis result that flavin binding to the enzyme results ina significant positive shift in midpoint potential. Howev-er, the disparity in the anthraquinone-2-sulfonate andbound flavin potentials was too large to allow accuratedetermination of the bound FAD potential. Analysisof data from titrations in which anthraquinone-(1,5)-disulfonate (Em7 = �174 mV) and indigo-(5,7)-disulfo-nate (Em7 = �125 mV) were used as solution potentialindicators allowed us to establish the midpoint potentialof bound FAD to be �149 ± 0.4 mV. Studies with afourth indicator, indigotrisulfonate (Em7 = �81 mV),resulted in the complete reduction of the indicator priorto flavin reduction. Absorbance spectra resulting fromthe reduction of FAD bound to N-terminally histi-dine-tagged SMOA in the presence of indigodisulfonateare presented in Fig. 6A. Fits through the data used tocalculate the potential of bound FAD and free FAD

Fig. 5. Single turnover kinetics of the reaction of N-terminally histidine-tagged SMOA with oxygen. Reduced 25–30 lM N-terminally histidine-tagged SMOA was reacted with 130 lM oxygen in 50% air-saturated buffer (d) and with mixtures of 130 lM oxygen and 500 lM styrene (s) orbenzene (n). Absorbance data superimposed with exponential fits are shown at (A) 314 nm (B), 390 nm, and (C) 450 nm. Extinction coefficientscorresponding to oxidized (m) and reduced (d) FAD–enzyme complexes and the first (s), second (j), and third (n) intermediates detected in thereaction of N-terminally histidine-tagged SMOA with (D) oxygen, (E) oxygen and styrene, and (F) oxygen and benzene.

Table 2Best-fitting values of rate constants in the oxygen reaction of SMOA

Reaction k1obs (s�1) k2obs (s

�1) k3obs (s�1) k4obs (s

�1)

Air only 4.7 ± 0.3 1.1 ± 0.1 0.30 ± 0.01 0.082 ± 0.004Air + benzene 5.0 ± 1.2 0.8 ± 0.2 0.032 ± 0.001 0.006 ± 0.01Air + styrene (15 ± 4.3)a 1.0 ± 0.2 0.02 ± 0.01 —

Absorbance data were fit to sums according to Eq. (4).a This value may be significantly influenced by the 21 ms time

constant associated with data averaging.


are shown in Fig. 6B. The oxidation–reduction potentialof free FAD was estimated at �212 ± 0.5 mV by analyz-ing the results of a reductive titration conducted in thepresence of the solution potential indicator, anthraqui-none-2-sulfonate, at 25 �C in 20 mM phosphate buffer(pH 7). There was no significant perturbation of themidpoint potentials of either FMN or riboflavin whenthey were reduced in the presence of N-terminally histi-dine-tagged SMOA. The ratio of the electron equilibri-um dissociation constants calculated from the freeFAD and SMOA-bound FAD equilibrium midpointpotentials indicates that N-terminally histidine-taggedSMOA binds reduced FAD 137 times more tightly thanoxidized FAD.

Mechanism of reduced FAD transfer from SMOB to

SMOA

The oxidation rate of reduced FAD in aerobic solu-tion is quite rapid and dependent on both the concentra-tion of oxygen and oxidized FAD present in thereaction. Kinetic traces from reactions of free reducedFAD with air-saturated buffer containing oxidizedFAD are shown overlaid with kinetic simulations of thisreaction in Fig. 7. The rate constants and model of re-duced flavin oxidation used in these simulations werebased on the known rate constants and kinetic mecha-nism previously reported for this reaction [7].

Clearly the presence of oxygen and oxidized flavinwill interfere with the efficiency of electron-transfer fromSMOB to SMOA if this transfer is mediated by diffusionof reduced flavin. The direct transfer of reduced flavinfrom SMOB to the active site of SMOA through the for-mation of transient complex of SMOA and SMOB dur-ing catalysis would eliminate interference caused byoxygen and oxidized FAD with the efficient transfer ofelectrons in SMO. To better resolve the nature of re-duced flavin transfer in this system, we measured thecoupling of NADH oxidation to styrene oxide produc-

Fig. 6. Equilibrium midpoint potential of FAD bound to N-terminally histidine-tagged SMOA. (A) Absorbance spectra from the dithionite-mediated reductive titration of 70 lM N-terminally histidine-tagged SMOA in complex with 34.7 lM FAD at 25 �C. The reaction was conducted in20 mM MOPSO buffer, pH 7, containing 200 lM styrene and 35 lM indigodisulfonate. (B) Plots of extent of reduction as a function of solutionpotential used to verify equilibrium midpoint potentials of free FAD (d) and FAD bound to N-terminally histidine-tagged SMOA (s).

Fig. 7. Reaction of free FAD with oxygen. Absorbance changes at450 nm corresponding to reactions of 6.3 lM reduced FAD (lowertrace) with 123 lM oxygen and 5.5 lM oxidized FAD and 8.3 lMreduced FAD (upper trace) with 123 lM oxygen and 3.5 lM oxidizedFAD in 20 mM phosphate buffer, pH 7, at 23 �C. Solid lines passingthrough the data represent simulations of each reaction.


tion as a function of FAD concentration. Results ofthese studies are shown in Fig. 8. The initial velocitydata describing the rate of NADH oxidation and styreneoxidation were recorded simultaneously as described inMaterials and methods. It is clear from inspection ofthese data and the NADH/styrene coupling ratio thatthe efficiency of reduced FAD transfer decreases withincreasing FAD concentration. Apparent Vmax and KMvalues for NADH reacting with SMOB of0.29 ± 0.1 lM s�1 and 4.8 ± 0.5 lM were calculatedfrom a Michaelis–Menten fit through the initial ratedata corresponding to the oxidation of NADH.

The styrene oxidation kinetics were fit in two ways. Inthe first approach, SMOB and SMOA were treated as an

enzyme complex and the data were fit to a modeldescribing uncompetitive substrate inhibition (Eq. (8)).Based on this fit, KM(app) (FAD) = 15 ± 8 lM and theinhibition constant for oxidized FAD is 37 ± 22 lM.

v ¼ V maxðappÞ½FADox�KMðappÞ þ ½FADox� 1þ ½FADox�K i

� � . ð8Þ

In the second approach to fitting these data, reducedFAD transfer was considered to occur exclusively by fla-vin diffusion. In this mode of operation, reduced flavin isa substrate of SMOA and in order to calculate theapparent KM of reduced FAD for SMOA it was neces-sary to estimate the concentration of reduced FAD con-tributing to each measurement of initial rate of styreneoxidation. This was done by inputting the apparentVmax and KM values for the reaction of SMOB withNADH into the integrated steady-state rate expression(Eq. (9)).

KNADHMðappÞV maxðappÞ

ln½NADH�o½NADH�t

� �þ ½NADH�o � ½NADH�t

V maxðappÞ

¼ t. ð9Þ

The average concentration of reduced FAD generatedover a 10-s time interval was determined in this wayfor each of the reactions of oxidized FAD with SMOB.The NADH/styrene coupling ratio calculated for eachreaction was then used to correct for the fraction of re-duced FAD consumed prior to interaction with SMOA.In this way, we obtained estimates of the reduced FADconcentration present in each of the initial rate determi-nations for the reaction of SMOA with styrene. The pro-gram GraphPad Prism 4 was then used to fit the styreneconcentration dependence data in Fig. 8 to a simpleproduct inhibition model (Eq. (10)).

Fig. 8. Coupling of NADH and styrene oxidation as a function ofFAD concentration. Initial rates of NADH (h) and styrene (s)oxidation superimposed with fits providing the best agreement with thedata as described in the text. Simulations of the experimentallydetermined NADH/styrene coupling ratio (j) according to Model A(dashed line) and Model B (dotted line) described in the text.

Fig. 9. Coupling of NADH and styrene oxidation as a function of N-terminally histidine-tagged SMOA/SMOB ratio. (A) Initial rates ofNADH (h) and styrene (s) oxidation catalyzed by various ratios ofSMOB and N-terminally histidine-tagged SMOA plotted together with(d) the NADH/styrene coupling ratio. Simulations of the experimen-tally determined NADH/styrene coupling ratio according to Model A(dashed line) and Model B (dotted line).


v ¼ V maxðappÞ½FADred�KMðappÞ 1þ ½FADox �K i

� �þ ½FADred�

. ð10Þ

In these fits, reduced FAD was treated as substrate andoxidized FAD as competitive inhibitor. The best esti-mates of the fit parameters were KM(app) (reducedFAD) = 1.3 ± 0.3 lM and Ki (oxidized FAD) = 96 ±68 lM. The equilibrium dissociation constant of re-duced FAD is calculated from the Ki and the observedshift in equilibrium midpoint potential of FAD uponbinding to SMOA was found to be �0.7 lM, whichis in close agreement with apparent KM for reducedFAD.

The coupling of NADH use to styrene oxidationwas further studied as a function of SMOA/SMOB ra-tio. In this investigation, the concentration of SMOAwas varied at fixed SMOB, FAD, NADH, and oxygenconcentration to yield SMOA:SMOB ratios varyingfrom 1:1 to 500:1. The observed initial rates of NADHand styrene oxidation are displayed in Fig. 8. A fitthrough the styrene oxidation data in accordance withthe Michaelis–Menten equation provides apparent val-ues of Vmax and KM of 0.095 ± 0.007 lM s

�1 and0.68 ± 0.16 lM.

Two models describing the dependence of SMO reac-tion on FAD concentration and enzyme ratio wereinvestigated by numerical simulation (Scheme 1). ModelA characterized by reactions 1–3 and 5 in Scheme 1 rep-resents flavin transfer from SMOB to SMOA by free dif-fusion. Model B is identical to Model A except that italso includes Scheme 1 reaction number 4. This modelrepresents a mechanism in which both direct and diffu-sive flavin transfer are significant features of catalysis.As illustrated, each model includes the synthesis of re-duced FAD by SMOB and the parallel pathways of fla-

vin reoxidation by either collision and reaction of freelydiffusing reduced flavin with molecular oxygen or theassociation of reduced flavin with SMOA followed itsreoxidation by the styrene epoxidation. Each model alsoincludes substrate inhibition of SMOA by oxidizedFAD in solution.

Parameters used in modeling these reactions were asfollows: the rate constants used to calculate the rate ofthe reaction of reduced flavin with oxygen and oxidizedflavin were based on the mechanism and values reportedin the literature [7]. The complex set of reactions in-volved in the reoxidation of free FAD is representedby k* in step 5 of Scheme 1. The relative amounts ofsuperoxide and hydrogen peroxide generated in thisreaction depend on the initial concentrations of oxygen,oxidized and reduced flavin. The rate of reduced flavinproduction by SMOB was based on our values of theapparent Vmax and KM of FAD corresponding to theconcentrations of oxidized FAD, NADH, and oxygenpresent in the reaction. The rate of styrene oxidationby free SMOA was modeled by using the calculated Kiof oxidized FAD and apparent Vmax and KM values ofreduced FAD were estimated from fits through the fla-vin concentration dependence of the reaction rate. Theresults from simulations of the FAD and SMOA con-centration dependence data according to Model A arecompared with the data in Fig. 8. The model correctlypredicts the general trends of increased substrate inhibi-tion of SMOA and decreased efficiency of couplingNADH consumption to styrene oxidation as a functionof increasing FAD concentration. Model A also correct-ly predicts the experimentally observed trend of in-creased coupling efficiency with increasing SMOA/SMOB ratio. Although Model A correctly predicts the

Scheme 1.


general trends observed in the experimental data, theestimates of coupling efficiency calculated from the sim-ulation are inconsistent with the experimentally ob-served values. At high FAD concentrations, thereaction is more than two orders of magnitude morecoupled than predicted by the simulation of the SMOreaction by Model A (Fig. 9). At low SMOA/SMOB ra-tios, the experimentally observed coupling of NADH tostyrene oxidation is about an order of magnitude greaterthan that calculated from the Model A simulation(Fig. 8).

In Model B, the channeling of FAD from SMOB toSMOA through the formation of a catalytic complexand the direct transfer of reduced flavin from SMOBto SMOA is predicted to decrease the fraction of re-duced flavin released in solution and the rate of free-fla-vin oxidation by reaction with molecular oxygen. Tostudy this model, it was necessary to estimate the mag-nitude of the dissociation equilibrium constant of thecomplex formed by SMOA and SMOB and the catalyticrate of styrene oxidation by this complex.

The sensitivity of the coupling efficiency to the mag-nitude of equilibrium dissociation constant of the com-plex formed between SMOA and SMOB duringcatalysis was estimated by comparing the experimentaldata to the results of a series of simulations in which thisvalue was varied in decades from 10 nM to 10 lM. Asimilar approach was taken in estimating the catalyticrate of styrene epoxidation by the SMOA–SMOB com-plex. Based on this evaluation, parameters giving thebest agreement with the experimentally determined datawere an SMOA–SMOB dissociation constant of 100 nMand observed catalytic rate constant of 10 s�1 for thestyrene oxidation reaction.

The results of this Model B simulation provided inFigs. 8 and 9 are in better agreement with the experi-mental data than those derived from the Model A. In

particular, the coupling efficiency predicted by ModelB is very close to the observed coupling efficiency ofthe SMO system over the full range of FAD and enzymeconcentrations investigated.

Discussion

The primary sequences of the flavin-dependent sty-rene monooxygenases are highly conserved[31,32,42,43]. The epoxidase components isolated fromPseudomonas S12 and VLB strains differ by only twoC-terminal residues and the flavin reductases of thesesystems have identical primary sequences [32].

Steady-state kinetic analysis of the reductase compo-nent from the VLB system was previously reported withFMN as substrate [15]. We find a different set of best-fit-ting kinetic parameters in our analysis of the reactionmechanism with FAD as the flavin substrate but areotherwise in agreement regarding the sequential natureof the reduction reaction mechanism.

The N-terminally histidine-tagged versions of SMOBand SMOA are more convenient to purify than the na-tive enzymes and appear to provide excellent functionalmodels for catalysis by the native enzyme components.As previously reported for the flavin reductase fromthe VLB system, we find that native SMOB expressespredominantly in the form of inclusion bodies, but thisprotein can be recovered in fully active form [15].

The N-terminally histidine-tagged version of SMOBsimilarly expresses primarily as insoluble protein andneither decreasing the temperature nor the concentra-tion of inducer during expression significantly improvesthe partitioning of SMOB into the soluble phase. How-ever, we were able to recover soluble highly purifiedN-terminally histidine-tagged SMOB constructs by Ni-affinity chromatography. The steady-state reactions of


native or N-terminally histidine-tagged SMOB with sub-strates and native or N-terminally tagged SMOA arevery similar.

Based on homology modeling through the 3dPSSMserver [44], the closest structural homology match forSMOB was an FMN-containing flavin reductase fromThermus thermophilis (Tahir Tahirov, unpublished re-sults). Other closely related structures are those ofFMN-dependent ferric ion and flavin reductases[45,46]. Homology modeling and model building studiesof the reductase of phenol hydroxylase reductase fromBacillus thermoglucosidasius suggest that this enzymemay also be a close structural relative of SMOB [14].

SMOA was most closely matched to p-hydrox-ybenzoate hydroxylase and D-amino acid oxidase bystructural homology modeling [47,48]. For this reason,we believe that the single-component flavin monooxyge-nases can provide valuable structural and mechanisticinsight into our studies of the oxidative half-reactionof SMO.

Reductive titrations of the SMOA–FAD complex inthe presence of 10–20 lM oxygen result in the formationof an extremely stable flavin intermediate. This specieshas an absorbance spectrum maximizing between 375and 380 nm, which is consistent with that of a C4a-flavinhydroperoxide. Interestingly, the intensity of this spec-trum was not diminished by the addition of excess dithi-onite. Upon mixing with styrene or excess oxygen thisspecies rapidly returns to that of oxidized flavin. Nooxygen intermediates were stabilized when the SMOA–FAD complex was titrated with dithionite in the pres-ence of 10–20 lM oxygen and 200 lM styrene. In thiscase, the equilibrium absorbance spectra recorded wererepresentative of mixtures of oxidized and two-electronreduced FAD.

In our rapid mixing studies of the reaction of SMOAwith oxygen, we observe the formation of the putativeflavin hydroperoxide intermediate at an observed rateconstant of 4.7 s�1 and calculate an apparent extinctioncoefficient of 11 mM�1 cm�1 at 380 nm for this species.In the presence of oxygen, this intermediate is trans-formed at an observed rate constant of 1 s�1 to a secondspecies with diminished extinction coefficient of8 mM�1 cm�1. Based on the difference spectrum result-ing from subtraction of the spectrum of the first interme-diate from that of the second, we conclude thatapproximately 20% of the bound FAD is reoxidized inthis phase of the reaction. Approximately 90% of thebound FAD returns to the oxidized form in a third stepwith an observed rate of 0.3 s�1. Enzyme-bound FAD isfully reoxidized in a final step with an observed rate con-stant of 0.08 s�1.

In the presence of the substrate analog benzene, thefirst intermediate again has an absorbance maximumin the 375–380 nm region and this species forms and de-cays with observed rate constants, which are very similar

to those found to provide the best fit in the reaction ofSMOA with oxygen in the absence of benzene. In thiscase, the first observed intermediate is spectrally less in-tense, having an extinction coefficient of only�8 mM�1 cm�1 at 380 nm. The enzyme returns to oxi-dized flavin with observed rate constants that are an or-der of magnitude less than those observed in the absenceof benzene. The rate of hydrolysis of the C4a-hydroxy-flavin has been shown to be sensitive to active site occu-pancy by substrate and products [49,50]. It seems likelythat the slow reoxidation of SMOA in the presence ofbenzene may similarly block the dissociation of hydro-gen peroxide and regeneration of oxidized flavin in theactive site of this enzyme.

When reduced SMOA is reacted with styrene, we ob-serve the rapid formation of an intermediate with anapparent molar extinction of 8 mM�1 cm�1 and absor-bance maximum centered at 370 nm. This intermediateforms at an observed rate of 15 s�1 then decays to yieldan oxidized FAD spectrum in a biphasic reaction. Eachdecay phase results in the formation of oxidized flavin.The first phase, which accounts for 90% of the increaseat 450 nm, occurs with an observed rate constant of0.95 s�1. A final increase in absorbance at 450 nm,which returns the fully oxidized FAD spectrum, occursat 0.02 s�1. In this experiment, the rate of styrene oxida-tion may have been limited by the reaction of oxygenwith the reduced FAD–SMOA. In this case, the C4a-hy-droperoxide intermediate would not accumulate and thefirst observed intermediate may be a C4a-hydroxyflavin.

The present data suggest the reactive intermediatesemployed in the oxidative half-reaction of SMOA aresimilar to those previously identified in related flavoen-zyme monooxygenases as C4a-peroxy and hydroxyflav-ins. However, many details of this reaction remain to beresolved. It is possible that the reactive species is actuallya flavin peroxide as has been established in the case ofcyclohexanone monooxygenase [51]. Alternatively, theflavin hydroperoxide may collapse to form a reactiveoxaziridine intermediate which then catalyzes the ob-served epoxidation [52] (Scheme 2).

Our titration data indicate that the equilibrium mid-point potential of SMOA-bound FAD is shifted posi-tively by 63 mV. This implies that the SMOA bindsreduced FAD approximately 137 times more tightlythan oxidized FAD (Scheme 3). Based on the best esti-mate of the FAD inhibition constant, we calculate thebinding constant of reduced FAD to SMOA is in therange of 500 nM. We presently have no data identifyingthe structural basis of this interaction, but the calculatedchange in binding energy �36 kJ mol�1 is comparablewith that which would be gained through the formationof a single hydrogen bond [53].

There was no evidence for the formation of a stableoxygen intermediate when either riboflavin or FMNwas substituted for FAD. Moreover, the midpoint

Scheme 2.

Scheme 3.


potentials of these FAD analogues were unaffected bySMOA based on reductive titrations, which includedthe solution potential indicators anthraquinone-2-sulfo-nate and indigodisulfonate. Our work supports the pre-vious finding for the VLB system that whereasriboflavin, FMN, and FAD each function as reductasesubstrates only FAD is an effective substrate for themonooxygenase component.

It was previously shown that the hydroxylase compo-nents of SMO (VLB) and the closely related 4-hydrox-yphenylacetate-3-monooxygenase system are able tocatalyze substrate oxygenation in the absence of theirreductase components [12,21]. It was further shownthrough gel filtration and analytical ultracentrifugationstudies that the reductase and monooxygenase compo-nents of these systems do not form stable complexes atequilibrium [15,20].

Additional studies of the 4-hydroxyphenylacetate-3-monooxygenase system were conducted to test for evi-dence of a reductase–hydroxylase interaction duringcatalysis. In this work, the apparent KM of the reductasefor FAD was measured and found to be uninfluenced bythe hydroxylase component [20]. A significant increaseor decrease in the apparent KM value would have pro-vided evidence in support of a direct reductase–hydrox-ylase interaction. However, this apparent KM valuedepends on a ratio of microscopic rate constants, whichmay increase, decrease, or not be significantly affectedby protein–protein interactions during catalysis, andthe absence of a significant change in this value doesnot preclude the formation of a transient reductase–hy-droxylase complex during catalysis.

In our studies of this problem in the SMO system, wedeveloped a real-time assay, which allows us to simulta-neously monitor the steady-state rates of styrene andNADH consumption. We find that within experimentaluncertainty NADH and styrene are oxidized in a 1:1 ra-tio only when the SMOA:SMOB ratio is large and whenFAD concentrations are kept low. Lower SMOA:-SMOB ratios and elevated FAD concentrations arefound to decrease the efficiency of this reaction.

To seek evidence for the formation of a transient fla-vin-transfer complex during catalysis by the SMO sys-tem, we compared our experimental data with theresults of numerical simulation of models, which includ-ed or excluded such a complex. Parameter estimates forthese simulations were derived from both our experi-mental data and from rate constants reported in the lit-erature for the reactions of free reduced flavin withoxygen. Based on this comparison, we find the observedsteady-state coupling of NADH and styrene oxidationto be in best agreement with a reduced flavin-transfermechanism in which SMOA and SMOB form a tran-sient complex during catalysis. In this way, the reducedflavin is protected from counterproductive reactionswith free oxygen and oxidized flavin in solution. Com-parison of the data with numerical simulations indicatesthat whereas the diffusion of free reduced flavin fromSMOB to SMOA is a significant mechanism of electrondelivery in the SMO system it does not account for theobserved reaction coupling efficiency over the broadrange of FAD concentrations and SMOA/SMOB ratiosinvestigated.

Flavin dynamics have been shown to be critical fea-ture in the catalysis of flavoenzyme monooxygenases[54]. In the two-component enzymes, this dynamicreaches an extreme in which the flavin is physicallytransferred from the reductase to hydroxylase. Since nei-ther FMN nor riboflavin is able to substitute for FAD inthe styrene epoxidation reaction, we propose that theAMP functionality represents a critical structural motifin the recognition and binding of FAD to SMOA duringcatalysis. Reduction of SMOA-bound FAD may becoupled to the movement of the isoalloxazine ring sys-tem from a solvent-exposed position to a sequesteredposition within the active site of the protein. This changeof environment is consistent with both the observed po-sitive shift in midpoint potential and the ability of theFAD to form a stable C4a-hydroperoxide.

The isoalloxazine ring of the oxidized FAD may besufficiently accessible such that SMOB can provide itwith electrons in the reductive half-reaction while theAMP portion of FAD is still associated with SMOA.On a similar note, the AMP moiety of FAD may remainsolvent exposed and accessible to SMOA during bindingand reduction of the isoalloxazine ring of free-FAD bySMOB. Based on the present information we suggestthat in the most efficient mode of catalysis by the

Scheme 4.


SMO system, SMOB may form a transient flavin-trans-fer complex with SMOA in which the AMP moiety isassociated with FAD and isoalloxazine ring is associatedwith SMOB (Scheme 4). Release of a tethered isoallox-azine ring from the active site of SMOB would ensureits efficient transfer to the oxygenation active site ofSMOA and minimize interaction with molecular oxygenand oxidized FAD in solution.

Acknowledgments

We thank the UCSF DNA-sequencing core facilityfor assistance in the verification of our cloned genesequences, Tom Franco in the San Francisco State Uni-versity College of Science and Engineering MachineShop for his help in constructing our stopped-flowinstrument, Drs. Ishan Erden (San Francisco State Uni-versity), David Ballou and Bruce Palfey (University ofMichigan), and Tahir Tahirov (RIKEN Institute) forhelpful discussions, NIH S06 GM52588 and for fundingthis research, and Robert Yen in the SFSU Mass Spec-trometry Core Facility supported by NIH P20MD000262, NCMHD for his help in verifying themolecular weight of SMOB.

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Mechanism of flavin transfer and oxygen activation by the two-component flavoenzyme styrene monooxygenaseMaterials and methodsCell growth, protein, and DNA purification from native P. putida S12Cloning design of expression vectors and sequencingExpression and purification of recombinant proteinsProtein concentration measurements and thiol titrationSteady-state reaction mechanism of SMOBPre-steady-state kinetic analysis of SMOAEfficiency of coupling NADH and styrene oxidation reactions of SMOOxidation ndash reduction potential measurements

ResultsProtein expression and purificationSteady-state mechanism of SMOBSteady-state analysis of SMOAPre-steady-state investigation of SMOAMeasurement of the Redox potential of FAD associated with SMOAMechanism of reduced FAD transfer from SMOB to SMOA

DiscussionAcknowledgmentsReferences

abb - san francisco state universityonline.sfsu.edu/gassner/chem897/smoa/pdf references/14.pdfabb...

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