Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA

JBC Papers in Press. Published on February 3, 2017 as Manuscript M116.774448

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.774448

Kinetic Mechanism of HadA monooxygenase

Kinetic Mechanism of the Dechlorinating Flavin-Dependent Monooxygenase

HadA

Panu Pimviriyakul¹, Kittisak Thotsaporn², Jeerus Sucharitakul²and Pimchai Chaiyen¹*

¹Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology,

Faculty of Science, Mahidol University, Bangkok, Thailand 10400.

²Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand 10300.

*Running title: Kinetic Mechanism of HadA monooxygenase

*Corresponding authors

E-mail: pimchai.cha@mahidol.ac.th. Phone: +66-2201 5596. Fax: +66-2354 7174.

Keywords: flavin-dependent monooxygenase, transient kinetics, oxygenase, dechlorinase, chlorophenols

(CPs).

ABSTRACT

The accumulation of chlorophenols (CPs)

in the environment, due to their wide use as

agrochemicals, has become serious

environmental problem. These organic halides can

be degraded by aerobic microorganisms, where the

initial steps of various biodegradation pathways

include an oxidative dechlorinating process in

the kinetic mechanism of a two-component flavin-

dependent monooxygenase that can catalyze

oxidative dechlorination of various CPs, and as

such will serve as the basis for future investigation

of enzyme variants that will be useful for

applications in detoxifying chemicals hazardous to

human health.

a

which chloride is replaced by

a hydroxyl

Chlorophenols (CPs) such as 2,4,6-

trichlorophenol (2,4,6-TCP), 2,4-dichlorophenol

(2,4-DCP) and pentachlorophenol (PCP) are

commonly used as herbicides, insecticides and

fungicides in agricultural industries. CPs can also

be derived from pesticide degradation. For

example, 2,4,5-trichlorophenol (2,4,5-TCP) is

derived from the degradation of 2,4,5-

trichlorophenoxyacetate (1), and 2-chloro-4-bromo

phenol is derived from the organophosphate

insecticide, profenofos (2). These compounds are

classified as persistent environmental pollutants

that can accumulate in water and soil (3), and most

of them are registered under the priority pollutants

list (4) and/or the priority list of hazardous

substances (5). High human exposure to these

compounds can lead to severe health problems such

as cancer, DNA damage, organ damage, or even

acute death (3,6-8). As these compounds are found

widespread in the environment due to their

utilization in agriculture and in industrial processes,

a biodegradation process that can provide a

sustainable method to detoxify them is essential to

the reduction of CP contamination in fresh water

and soil.

substituent. Harnessing these dechlorinating

processes could provide an opportunity for

environmental remediation, but detailed catalytic

mechanisms for these enzymes are not yet known.

To close this gap, we now report transient kinetics

and product analysis of the dechlorinating flavin-

dependent monooxygenase, HadA, from the

aerobic organism Ralstonia pickettii DTP0602,

identifying several mechanistic properties that

differ from other enzymes in the same class. We

first overexpressed and purified HadA to

homogeneity. Analyses of the products from single

and multiple turnover reactions demonstrated that

HadA prefers 4-CP and 2-CP over CPs with

multiple substituents. Stopped-flow and rapid-

quench flow experiments of HadA with 4-CP show

the involvement of specific intermediates (C4a-

hydroperoxy-FAD and C4a-hydroxy-FAD) in the

reaction, define rate constants and the order of

substrate binding, and demonstrate that the

hydroxylation step occurs prior to chloride

elimination. The data also identify the non-

productive and productive paths of the HadA

reactions and demonstrate that product formation is

the rate-limiting step. This is the first elucidation of

1

Kinetic Mechanism of HadA monooxygenase

Many aerobic biological systems have

shown promising activity in degrading CPs.

Degradation of CPs such as 2,4,5-TCP, 2,4,6-TCP,

or PCP can be carried out by various enzymatic

systems such as tft clusters from Burkholderia

cepacia AC1100 (9,10), tcp clusters from Ralstonia

eutropha JMP134 (11-13), had clusters from

Ralstonia pickettii DTP0602 (14-16) and pcp

clusters from Sphingobium chlorophenolicum (17-

19).

detoxification. Previously, studies of the HadA

reaction were only carried out with whole cells and

lysates of cells overexpressing HadA (14,15)

without measuring the products that were

specifically generated from the flavin-bound

enzyme. As study of the transient kinetics of a two-

component flavin-dependent dechlorinase has not

been reported, we have chosen HadA as a model for

investigating the substrate utilization and rate

constants associated with the individual steps under

single turnover conditions to identify the flavin

species and reaction steps and pathways involved in

oxidative dechlorination.

Aerobic biodegradation of CPs generally

involves replacement of a chloride group by a

hydroxyl group in the initial steps of these

biodegradation

dehalogenation is often catalyzed by flavin-

dependent monooxygenases (20). These

oxygenases include both types of monooxygenases-

those that utilize a single protein, as well as those

that utilize two proteins. The most well studied

single-component flavin-dependent dechlorinase is

PcpB from S. chlorophenolicum. This enzyme

binds to FAD as a cofactor and requires NADPH

and O₂as co-substrates for the conversion of PCP

pathways.

This

oxidative

Based on sequence similarity, HadA

belongs to the family of two-component flavin-

dependent monooxygenases that utilize two

separate proteins in their catalysis (Class D

monooxygenase) (22). A reductase component of

these enzymes catalyzes flavin reduction, while an

oxygenase

component

catalyzes

substrate

oxygenation (22,23). The most well-studied

enzyme in this family is p-hydroxyphenylacetate

hydroxylase

(HPAH)

from

Acinetobacter

to

2,3,5,6-tetrachlorobenzoquinone

(2,3,5,6-

baumannii. The mechanism of reduced flavin

generation by the reductase (C₁), as well as the

mechanism of the oxygenase (C₂) reaction, have

been determined for this enzyme. Furthermore, the

individual rate constants associated with each step

and the mechanism of reduced flavin transfer

between the two components have been reported

(24-30). This knowledge has been useful for

applying HPAH for the synthesis of bioactive

phenolic acids and in the hydroxylation of aniline

derivatives (31,32).

TCBQ) in the initial step of PCP degradation. The

quinone product can be chemically reduced to

2,3,5,6-tetrachloro-p-hydoquinone

(2,3,5,6-

TCHQ) (18,19). However, turnover of PcpB using

PCP as a substrate is very slow, implying that PCP

may not be a native substrate of PcpB (18). Many

oxidative dechlorination reactions are also

catalyzed by two-component flavin-dependent

enzymes including TftD from B. cepacia AC1100

(9,10), TcpA from R. eutropha JMP134 (13), and

HadA from R. pickettii DTP0602 (15). Although a

few of these enzymes have been shown to catalyze

oxidative dechlorination of CPs, their reaction

mechanisms are not well understood. None of these

enzymes have been explored with regards to their

kinetic mechanisms, and the rate constants

associated with the individual steps have yet to be

measured. Only X-ray structures of TftD and TcpA

and the overall bioconversion activities of these

enzymes have been reported (9,10,13,15,21).

When the sequence of HadA was examined

by sequence similarity network (SSN) analysis

using the Enzyme Function Initiative (EFI)

program to determine its relationship with any

homologous enzymes known to date, the results

(Fig. S1) showed that there are 2,839 enzymes in

which the sequences have >34% identity with

HadA and only 11 out of these 2,839 enzymes were

reviewed by UniProt. The enzymes identified to be

homologous to HadA were mostly flavin-

dependent monooxygenases that incorporate a

hydroxyl group into phenols such as CPs or

nitrophenols (9,10,33-35). As these enzymes

showed potential applications in bioremediation of

toxic compounds, the understanding of kinetic

mechanisms associated with a flavin-dependent

Based on previous reports, the substrate

utilization of HadA (14,15) seems to be broader

than that of TcpA (specific to 2,4,6-TCP; 86.7%

identity to HadA) and TftD (specific for 2,4,5-TCP;

64.2% identity to HadA), making HadA attractive

for development as

a

biocatalyst for CP

2

Kinetic Mechanism of HadA monooxygenase

dechlorinase (HadA) reported herein should also be

useful for giving an overall picture about the

reaction mechanism of these enzymes in general.

S4A). In the presence of HadA, the oxidation of

FADH^-resulted in two observable phases. The first

phase consisted of a large absorbance increase at

380 nm (blue solid line, Fig. S4A), while the second

phase was characterized by a large and slow

(observed rate constant of 0.005 s^-1) increase in

absorbance at 450 nm (blue dashed line, Fig. S4A).

These data indicate that in the presence of HadA,

FADH^-oxidation resulted in the formation of C4a-

hydroperoxy-FAD which can be detected by a large

absorbance increase at 380 nm where C4a-

hydroperoxy-FAD absorbs maximally (more

analysis to follow). The mixing of FMNH^-with air-

saturated HadA (blue lines, Fig. S4B) yielded the

same kinetic traces as those of the reaction without

HadA (red lines, Fig. S4B). Both reactions showed

the increase in absorbance at 380 and 446 nm with

an observed rate constant of 3.2 s^-1. These data

clearly suggest that HadA can only bind to FADH^-

and not FMNH^-.

In this work, the enzyme from R. pickettii

DTP0602 (HadA) was overexpressed, purified to

homogeneity, and its reaction with several CPs

were explored. The reactions of HadA with 4-

chlorophenol (4-CP, the most optimal substrate)

were investigated using stopped-flow and rapid

quench-flow techniques to measure the rate

constants associated with the individual steps. The

results clearly determined the order of substrate

binding and the rate constants associated with each

of the individual steps, and identified C4a-

hydroperoxy-FAD and C4a-hydroxy-FAD as

intermediates in the dechlorination process

catalyzed by HadA.

RESULTS

Expression

and

purification

of

HadA.

Recombinant HadA was overexpressed and

purified according to the purification methods

described in the Experimental Procedures to yield

protein with >95% purity (Fig. S2). The protein

preparation protocol resulted in ~0.6 g of purified

HadA from 3.9 l of culture. HadA had no cofactor

bound and the native molecular mass suggests that

HadA is a tetrameric protein with a subunit

molecular mass of ~59 kDa (Fig. S3).

Identification of product from HadA single

turnover reactions. In order to identify the

substrate specificity of HadA, the products

resulting from single turnover reactions of HadA

(see Experimental Procedures) were identified by

comparing their HPLC and mass spectroscopic

profiles with those of standard compounds. As most

of the products were expected to be quinone

derivatives, ascorbic acid (1 mM) was added into

the reaction to convert them into their

hydroquinone form which is more stable. The

results of the product identification studies (Table

1, Figs. S5A-S5C) show that HadA catalyzes the

hydroxylation of various CPs including 4-

chlorophenol (4-CP), 2-chlorophenol (2-CP), 2,4-

dichlorophenol (2,4-DCP), 2,4,5-trichlorophenol

(2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP)

Flavin specificity of HadA. As HadA is the

oxygenase component of a two-component flavin-

dependent monooxygenase, we first needed to

identify its flavin specificity and its ability to form

C4a-hydroperoxyflavin. The enzymes in this class

use specific reduced flavins as substrates and form

C4a-hydroperoxyflavin as a reactive intermediate

to perform monooxygenation plus other additional

activities (22,23,36,37). Binding of FADH^-or

FMNH^-to HadA was investigated to identify which

flavin could bind to HadA and yield a binary

complex able to react with oxygen. Solutions of

FADH^-and FMNH^-were mixed with buffers

containing oxygen (air saturation) in the presence

or absence of HadA. The results indicate that only

the oxidation of FADH^-was affected by the

presence of HadA, indicating that the enzyme can

only bind to FADH^-and not FMNH^-(Fig. S4). The

reaction of free FADH^-with oxygen showed an

increase in absorbance at 380 and 450 nm with an

observed rate constant of ~2.3 s^-1(red lines, Fig.

to

produce

hydroquinone

(CHQ),

(HQ),

2,5-

chlorohydroquinone

dichlorohydroquinone (2,5-DCHQ), and 2,6-

dichlorohydroquinone (2,6-DCHQ), respectively.

The product identification results are summarized

in Table 1. Based on these reactions, HadA

incorporates a hydroxyl group at the para-position

of the original hydroxyl group of all five substrates

investigated. When the para-position contains a

chloride substituent (in the case of 4-CP, 2,4-DCP,

2,4,5-TCP and 2,4,6-TCP), HadA catalyzes

hydroxylation plus Cl^-elimination (Fig. 1).

Comparison of the percentage of product formation

3

Kinetic Mechanism of HadA monooxygenase

after single turnover reactions for the different

substrates indicates that HadA prefers to use mono-

substituted CPs over multiple-substituted CPs.

These results contradict previously reported data

from cell lysate experiments (where HadA was not

purified) in which 2,4,6-TCP was reported as the

most preferred substrate for HadA (14,15).

position 4 and then the second hydroxylation and

Cl^-elimination step proceeds at position 2.

pH optimization of HadA. In order to identify the

optimum pH for the HadA reaction to be used in

transient kinetics experiments, single turnover

reactions of HadA and its best substrate, 4-CP were

carried out at various pHs and the amount of

product formation was analyzed according to the

protocol described in the Experimental Procedures.

An anaerobic solution of HadA:FADH^-in 20 mM

HEPES pH 7.5 was mixed with an equal volume of

an aerobic solution of 4-CP at various pH values

from 6.5-10.5. The HQ production results (Fig. 4)

indicated that the reaction of HadA has an optimum

pH within the range of ~7.0-8.2. Therefore, the

transient kinetics experiments were all performed at

pH 7.5. (Transient kinetic experiments were also

performed at various pHs to explore if pH affects

the ability of HadA to form C4a-hydroperoxy-FAD

(shown in Section. F).

Comparison of multiple turnover reactions of

HadA with various chlorophenols. As the results

from the single turnover experiments in Table 1 are

different from those previously reported (14,15),

multiple turnover reactions of HadA (see

Experimental Procedures) with various substrates

were carried out to identify which compounds

among 4-CP, 2,4-DCP, 2,4,5-TCP, and 2,4,6-TCP,

are the preferred substrates for the HadA

bioconversion process. The results (Fig. 2) show

that under the same experimental conditions, 4-CP,

2,4-DCP, and 2,4,5-TCP were completely

converted to HQ, CHQ, and 2,5-DCHQ with half-

times (t_1/2) of the reactions around 13, 35, 56 min,

respectively (Figs. 2A-C). In contrast, 2,4,6-TCP

was only 72% depleted after 600 min (Fig. 2D).

These data agree with the results from single

turnover reactions (Table 1) which indicated that

the efficiency of HadA hydroxylation of 4-CP

(49%) is greater than that of 2,4-DCP (14%), 2,4,5-

TCP (9%), and 2,4,6-TCP (4%).

Effect of ascorbic acid on the HadA reaction. As

most of the reactions in this report contained

ascorbic acid (1 mM) to stabilize the hydroquinone

products, the effect of ascorbic acid on the reaction

of HadA was investigated. The transient kinetics of

the HadA reactions in the absence or presence of

ascorbic acid was investigated using stopped-flow

techniques. A solution of the HadA:FADH^-binary

complex was mixed with air-saturated 20 mM

HEPES pH 7.5 in the presence or absence of 1 mM

Preference of hydroxylation site identified by

multiple turnover reactions of HadA and 2,4,5-

TCP. The results in Fig. 2C also show that the

product from 2,4,5-TCP multiple turnover

reactions, 2,5-DCHQ, decreased over time,

indicating that the compound may be further

hydroxylated. To characterize the nature of the

second conversion step of 2,4,5-TCP, the reaction

of HadA with 2,5-DCHQ was investigated. The

results showed that 2,5-DCHQ was completely

depleted within 4 hrs (Fig. S6A), with concomitant

appearance of a hydroxylation product which has

an m/z of +16 atomic units compared to the value

for 2,5-DCHQ. This compound also has mass

spectroscopic and HPLC profiles consistent with

those of 5-chlorohydroxyquinol (5-CHQL) (Figs.

S6A-B). All results suggest that HadA can convert

2,5-DCHQ to 5-CHQL. Therefore, the overall

reaction of HadA and 2,4,5-TCP can be

summarized as in Fig. 3 in which the first

hydroxylation and Cl^-elimination step occurs at

ascorbic

acid

on

the

stopped-flow

spectrophotometer. Kinetic traces of both

experiments were almost identical (Fig. S7),

suggesting that ascorbic acid does not interfere with

HadA catalytic behavior and that it can be used for

stabilizing the substrate and product in the HadA

reaction.

Reaction mechanism of HadA with 4-CP as a

substrate investigated by transient kinetics.

A. Binding of FADH^-. (Step 1 in Fig. 5; FADH^-is

the first substrate to bind to HadA)

For two-component flavin-dependent

monooxygenases that use reduced flavin as a

substrate such as HadA, the enzyme must bind to

FADH^-to form an active binary complex

HadA:FADH^-before it can react with oxygen to

form C4a-hydroperoxy-FAD.

4

Kinetic Mechanism of HadA monooxygenase

A.1. Dissociation constant (K_d) of binding of HadA

and FADH^-.

HadA:FADH^-binary complex results in formation

of C4a-hydroperoxy-FAD which eliminates H₂O₂

slower than the oxidation of free FADH^-(Section

A.1)). Results (Fig. S8A) showed that the behavior

of free FADH^-oxidation only disappeared when the

incubation time of the first mixing was longer than

20 s. A plot of the absorbance changes at 450 nm at

2 s (representing the amount of HadA:FADH^-

binary complex formed) versus the incubation age

time was analyzed according to a single exponential

equation to yield an observed rate constant of 0.33

± 0.04 s^-1which is equivalent to a bimolecular rate

constant for the binding of HadA and FADH^-of 4.4

± 0.5 × 10³M^-1.s^-1(inset in Fig. S8A).

The K_dfor the binding of HadA and FADH^-

can be measured based on the amount of the binary

complex formed which can further react with

oxygen to form C4a-hydroperoxy-FAD (Fig. S4).

A solution of anaerobic FADH^-(12.5 µM) was

mixed with air saturated HadA (0-0.4 mM) in the

stopped-flow apparatus. The concentrations

indicated are final concentrations after mixing. The

formation of the HadA:FADH^-binary complex is

reflected by a slower rate of flavin oxidation (which

occurs upon H₂O₂elimination from C4a-

hydroperoxy-FAD, Fig. S4A; Step 8 in Fig. 5) with

a t_1/2~100 s (solid lines, Fig. 6). This is much slower

than the oxidation rate of free FADH^-(t_1/2~ 0.3 s,

dashed line in Fig. 6). When the binding of HadA

and FADH^-was not complete, the traces showed

two observable phases that directly reflected the

oxidation of two distinct species of free and HadA-

bound FADH^-(Fig. 6). Therefore, at t=2s, the

difference between the absorbance at 450 nm of

free FADH^-and that of the HadA-bound FADH^-

oxidation represents the amount of HadA:FADH^-

binary complex formed. A plot of these values

versus free HadA concentration represents a

binding isotherm that is consistent with the K_dvalue

of 2.0 ± 0.2 µM (inset on Fig. 6). This result also

established the experimental conditions required to

achieve complete binding of FADH^-by HadA (a

ratio of HadA:FADH^-as 3:1).

B: Formation of C4a-hydroperoxyflavin in the

HadA reaction.

B.1. Nature of C4a-hydroperoxy-FAD formation.

(Step 3 in Fig. 5)

To obtain a spectrum of C4a-hydroperoxy-

FAD and to characterize the kinetics of the reaction,

a solution of the HadA:FADH^-binary complex was

mixed with various concentrations of oxygen in 20

mM HEPES pH 7.5. Analysis of the results (upper

inset in Fig. 7) indicates that kinetics of these

reactions are composed of three phases. The first

(0.01-0.2 s) and second (0.2-6 s) phases showed

large increases in the absorbance at 380 nm without

significant changes at 450 nm. Both phases were

dependent on the oxygen concentration. Plots of the

observed rate constants (k_obs) of both phases versus

oxygen concentrations were linear, consistent with

second-order rate constants of 6.4 ± 0.4 × 10⁴M^-1.s^-

¹and 3.3 ± 0.5 × 10³M^-1.s^-1(lower inset in Fig. 7),

respectively. The dependence of both phases on

A.2 Kinetics of HadA and FADH^-binding. (Step 1

in Fig. 5)

The binding rate of FADH^-to HadA was

measured using double-mixing stopped-flow

experiments by monitoring the progress of

HadA:FADH^-formation. An anaerobic solution of

FADH^-(25 µM) was mixed with an anaerobic

solution of HadA (75 µM) in the first mixing step

and incubated for various periods of time (0.01-200

s) before oxygenated buffer (air saturation) was

added in the second mixing step. Kinetic traces

were obtained by monitoring the change of

absorbance at 450 nm. A longer preincubation time

prior to adding the oxygenated buffer resulted in

slower kinetics of absorbance 450 nm increase,

raising the t_1/2from ~0.3 s (free FADH^-oxidation)

to ~100 s (HadA-bound FADH^-reaction). This is

because formation of the HadA:FADH^-complex is

more complete (as explained above, the

oxygen

concentration

suggests

that

the

HadA:FADH^-complex probably exists as a mixture

of fast and slow reacting species (Step 2 in Fig. 5).

These two phases represent the reaction of the

HadA:FADH^-complex with oxygen to form C4a-

hydroperoxy-FAD because the spectrum at the end

of each phase (solid line with filled circles in Fig.

7) is similar to the spectrum of C4a-

hydroperoxyflavin that is produced in the reactions

of

the

oxygenase

component

of

p-

hydroxyphenylacetate hydroxylase (25) and

luciferase (38). The last phase showed a decrease in

absorbance at 380 nm and a large increase in

absorbance at 450 nm. The observed rate constant

was independent of oxygen concentration. This

phase represents a slow intermediate decay by H₂O₂

5

Kinetic Mechanism of HadA monooxygenase

elimination with a rate constant of 5.4 ± 0.2 × 10^-3

s^-1to form the final species, oxidized FAD (Step 8

in Fig. 5). These data indicate that in the absence of

an aromatic substrate, the overall reaction of the

binary complex of HadA:FADH^-with oxygen can

be summarized as in Steps 3 and 8 in Fig. 5.

C. Binding of 4-CP and HadA. (Step 10 in Fig. 5,

4-CP can bind to HadA but does not lead to a

productive path.)

C.1. 4-CP can also bind to apoHadA and this

binding prevents the binding of FADH^-.

Although formation of the HadA:FADH^-

binary complex in a two-component flavin-

dependent monooxygenase is required before the

enzyme can react with oxygen, it is known that

certain substrates can also bind to some enzymes to

form a dead-end complex before the binding of

reduced flavin (38-40). To identify if 4-CP can bind

to apoHadA, solutions of air saturated HadA (75

µM) that were pre-incubated with various

concentrations of 4-CP (0.1–10 mM) were mixed

with an anaerobic solution of FADH^-(25 µM) to

monitor the formation of C4a-hydroperoxy-FAD

using a stopped-flow spectrophotometer. Final

concentrations are given as after mixing. If a

complex of HadA:4-CP can form, the reaction of

enzyme-bound FADH^-and oxygen would react

differently from the reaction in the absence of 4-CP.

Therefore, the kinetic traces of the reactions were

measured at 380 (Fig. 8A) nm and 450 nm (Fig. 8B)

to monitor whether or not the presence of 4-CP

perturbs the formation of C4a-hydroperoxy-FAD.

The results clearly show that upon increasing the 4-

CP concentration, the amount of C4a-hydroperoxy-

FAD formed decreased, and the kinetics of the

reaction became more similar to that of the reaction

of free FADH^-with oxygen (not bound to HadA).

At the highest 4-CP concentration used (orange line

in Figs. 8A-B), the reaction was practically the

same with the reaction of free FADH^-reacting with

oxygen (dashed line in Figs. 8A-B). These data

clearly demonstrate that 4-CP can bind to HadA and

this binding prevents the binding of FADH^-to

HadA i.e. 4-CP can bind to HadA to form a dead-

end complex that cannot proceed through a

productive path (Step 10 in Fig. 5). In another case

in which HadA was pre-incubated with FADH^-to

form the HadA:FADH^-binary complex prior to

mixing with 4-CP (0.1–10 mM), it was found that

at concentrations of 4-CP <2 mM, C4a-

hydroperoxy-FAD can still form to the same extent

as in the reaction in the absence of 4-CP (Fig. S9A).

This implies that once the HadA:FADH^-complex is

formed, 4-CP cannot compete off the FADH^-

binding on HadA at concentrations <2 mM.

B.2. Rate of a conformational change between two

forms of the HadA:FADH^-binary complex. (Step 2

in Fig. 5)

We noted that a mixing of an aerobic HadA

solution with an anaerobic FADH^-solution resulted

in C4a-hydroperoxy-FAD formation with

a

monophasic kinetic trace (Fig. S4A) which is

different from the results in the upper inset of Fig.

7

that shows biphasic formation of C4a-

hydroperoxy-FAD. These results suggest that pre-

incubation of HadA and FADH^-may lead to a

conformational change in the enzyme, in which one

form of the HadA:FADH^-complex reacts faster

with oxygen than the other. To determine whether

the pre-incubation of HadA with FADH^-did indeed

cause this effect, double-mixing stopped-flow

experiments similar to those in Section A.2 were

carried out in which the incubation time of the first

mixing step to form the HadA:FADH^-complex was

varied. In the second mixing step, an oxygenated

solution was added to form C4a-hydroperoxy-

FAD, which could then be detected at 380 nm (Fig.

S8B). The results in Fig. S8B indicate that at an

incubation time of <20 s, the kinetics of C4a-

hydroperoxy-FAD formation was monophasic with

a bimolecular constant of 9.9 ± 0.1 × 10⁴M^-1.s^-1.

When the incubation time was increased to greater

than 20 s, biphasic kinetics of C4a-hydroperoxy-

FAD formation was observed. These results

indicate that the biphasic kinetics observed when

mixing

a

preformed binary complex of

HadA:FADH^-with oxygen was indeed due to a

conformational change in the enzyme to result in

two forms of the complex, HadA:FADH^-(fast

reacting species) and HadA*:FADH^-(slow reacting

species). The rate constant for this enzymatic

conformational change was calculated from a plot

of ΔA380 nm at t=2 s (the decrease in absorbance

at 380 represents conversion of the enzyme to the

slow reacting species) versus the incubation time as

0.14 ± 0.01 s^-1(inset in Fig. S8B).

6

Kinetic Mechanism of HadA monooxygenase

C.2. Formation of 4-CP and HadA leads to

formation of a dead-end complex that does not

result in dechlorination.

against the 4-CP concentration to calculate k_on, k_off

and K_dfor the binding of 4-CP to HadA as 25 ± 2

M^-1.s^-1, 10 ± 2 × 10^-4s^-1, and 40 ± 10 µM,

respectively (Fig. S10B).

In order to confirm that formation of the

HadA:4-CP complex (in section C.1) is not relevant

to a productive path with dechlorination activity,

the amounts of product (HQ) formed from two

different mixing experiments in which HadA:4-CP

was preformed, or not preformed (Figs. 8A-B and

Fig. S9) were measured and compared. The results

(inset in Fig. 8A) clearly show that higher amounts

of 4-CP (>2 mM) reduced the amount of product

formation in both mixing methods, indicating that

4-CP exerts a substrate inhibition effect. However,

mixing of a preformed complex of HadA:4-CP plus

oxygen with FADH^-resulted in a much lower

amount of product formation than the mixing of a

binary complex of HadA:FADH^-with oxygen plus

4-CP (inset in Fig. 8A). At 4-CP concentrations

>3.2 mM, the preformed complex of HadA:4-CP

did not give any dechlorinated product. This result

also agrees with the kinetic traces because at this

concentration of 4-CP, FADH^-could not bind to

HadA and only free FADH^-oxidation was observed

(orange line in Figs. 8A-B). All data suggest that 4-

CP can bind to apoHadA to form a dead-end

complex that does not lead to dechlorination (Step

10 in Fig. 5).

A dissociation constant (K_d) for the binding

of HadA and 4-CP could also be calculated from the

amount of free FADH^-in the system because the

amount of free FADH^-is inversely proportional to

the amount of 4-CP occupying HadA. Analysis of

the K_dvalue was carried out according to Equation

S5 (Supplemental Information). Upon increasing

the 4-CP concentration, more free FADH^-oxidation

which is oxidized faster than the HadA-bound

FADH^-(Fig. 8B) was observed. Therefore, the

increase of absorbance at 450 nm at 2 s (which is

due to free FADH^-oxidation) with increasing 4-CP

was used for calculating the K_dfor the binding of

HadA and 4-CP as 29 ± 4 µM (inset in Fig. 8B).

This value agrees well with the dissociation

constant obtained from the kinetics experiments

mentioned above.

D. 4-CP binds to HadA after C4a-hydroperoxy-

FAD is formed in the productive path. (Step 4 in

Fig. 5).

The results in sections A-C indicate that

FADH^-is the first substrate to bind to HadA.

Although 4-CP can bind to HadA, the HadA:4-CP

is a dead-end complex that does not lead to

formation of C4a-hydroperoxy-FAD. In order to

proceed with the productive path, 4-CP must bind

the HadA:FADH^-complex or the HadA:C4a-

hydroperoxy-FAD intermediate. To identify if 4-

CP can bind to a binary complex of HadA:FADH^-

before it reacts with oxygen, two stopped-flow

experiments were carried out to monitor rate

constants of C4a-hydroperoxyFAD formation and

decay. In the first experiment, a solution of

HadA:FADH^-in the presence of 4-CP was mixed

with aerobic buffer while in the second experiment,

a solution of HadA:FADH^-binary complex alone

was mixed with aerobic buffer plus 4-CP. Both

experiments were monitored for absorbance

changes at 380 and 450 nm to observe the formation

of C4a-hydroperoxy-FAD. If the ternary complex

can form, it likely reacts with oxygen differently

from the reaction of the binary complex of

HadA:FADH^-with oxygen. Previous studies of C₂

oxygenase, which is also a two-component

oxygenase, have shown that a ternary complex of

C₂:FMNH^-:HPA reacts with oxygen ~50-fold

C.3 Binding kinetics and dissociation constant of

the HadA:CP complex.

The rate of 4-CP binding to HadA was

investigated using double-mixing stopped flow

experiments. In the first mixing, an aerobic solution

of HadA (75 µM) was mixed with 4-CP (0.8, 1.6,

3.2, and 6.4 mM) and was incubated at various time

periods before the solution was mixed against an

anaerobic solution of FADH^-(25 µM) in the second

mixing. Concentrations indicated are final

concentrations. Longer incubation times and higher

4-CP concentrations allowed more complete

formation of the HadA:4-CP complex, leading to

the prevention of FADH^-binding and the resulting

free FADH^-is oxidized with t_1/2~0.3 s. Therefore,

the progress of 4-CP binding to HadA can be

monitored by plotting ΔA450 nm at t=2 s versus

incubation times of the first mixing. The plots were

analyzed using an exponential equation to calculate

the observed rate constant of HadA and 4-CP

binding at each 4-CP concentration (Fig. S10A).

The observed rate constant values were plotted

7

Kinetic Mechanism of HadA monooxygenase

slower than the reaction of a binary complex of

C₂:FMNH^-(25). The results (Fig. S11) show similar

kinetic traces for both experiments, indicating that

pre-incubation of 4-CP with the HadA:FADH^-

binary complex had no influence on the rate

constant of C4a-hydroperoxy-FAD formation.

These results suggest that the a ternary complex of

HadA:FADH^-:4:CP is not necessary and that 4-CP

likely binds to the enzyme after C4a-hydroperoxy-

FAD formation (Step 4 in Fig. 5).

significantly different from those data of the

reaction in the absence of substrate. The rate

constant of the reaction in the presence of 4-CP was

6.9 ± 0.2 × 10^-2s^-1, ~13 fold faster than that without

substrate (5.4 ± 0.2 × 10^-3s^-1). Although the kinetics

showed only a single observable phase, this third

phase is the combination of the reactions of a

coupling

path

in

which

dechlorination

(hydroxylation and Cl^-elimination) takes place

(Steps 4-7 in Fig. 5) and an uncoupling path in

which oxidized FAD is produced by H₂O₂

elimination (Steps 8&9 in Fig. 5). In order to obtain

the individual rate constants associated with each

reaction path, stopped-flow spectrofluorometric

(Section E.2) and rapid-quench flow (Section E.3)

experiments were carried out.

E. Kinetics of HadA:FADH^-binary complex

reaction with O₂in the presence 4-CP.

The results in Sections A-D have

established the kinetic mechanism for the reaction

of HadA from Steps 1-4 & 10 in Fig. 5. The

knowledge obtained was used for setting up a

mixing condition that allows the reaction of HadA

to proceed in a productive manner by avoiding pre-

mixing of HadA and 4-CP to prevent formation of

the dead-end complex (Step 10 in Fig. 5) and by

using HadA:FADH^-with a ratio of 3:1 to allow for

complete formation of an active HadA:FADH^-

binary complex.

E.2. Detection of the hydroxylation rate constant by

monitoring formation of the C4a-hydroxy-FAD

intermediate

using

stopped-flow

spectrofluorometry.

As the hydroxylation of 4-CP to form HQ

requires the transfer of the hydroxyl group from

C4a-hydroperoxy-FAD to the substrate to result in

C4a-hydroxy-FAD, monitoring the kinetics of the

change between the two forms of the flavin

intermediates would be a direct measurement of the

rate constant associated with hydroxyl group

transfer from C4a-hydroperoxy-FAD into 4-CP.

Previous studies have shown that C4a-

hydroperoxyflavin and C4a-hydroxyflavin in some

enzymes have different fluorescence characteristics

(36,37,41-43), permitting direct measurement of

the hydroxylation step. Therefore, an experiment

similar to Section E.1 was carried out but the flavin

fluorescence was monitored rather than

absorbance. The fluorescence kinetics of the

reactions in the presence of 4-CP was monitored by

using excitation wavelengths at 380 nm (Ex380)

and 450 nm (Ex450) to monitor emission of C4a-

hydroxy-FAD and oxidized FAD, respectively.

Both flavin species were detected by their

fluorescence at emission wavelengths >495 nm

(Em >495). The results showed three observable

phases (red lines in Fig. 9). The first phase (0.001-

0.2 s) is a lag phase with a very small change of

fluorescence signal at both excitation wavelengths.

This phase occurs at the same time as the formation

of HadA:C4a hydroperoxy-FAD monitored by

absorbance. These data indicate that HadA:C4a-

hydroperoxy-FAD is not highly fluorescent. The

E.1. Reaction of the HadA:FADH^-binary complex

with O₂in the presence of 4-CP monitored by

stopped-flow spectrophotometry.

A solution of HadA:FADH^-(75 µM:25

µM) binary complex was mixed against buffer

solutions containing 4-CP (0.8 mM) and various

concentrations of oxygen (0.13, 0.31, and 0.61 mM)

in 20 mM HEPES pH 7.5 at 25^oC in the stopped-

flow spectrophotometer. Concentrations indicated

are given as final concentrations. Changes in

absorbance at 380 and 450 nm were recorded to

monitor the formation and decay of C4a-adduct

intermediates and formation of the final species of

oxidized FAD, respectively. The reaction in the

presence of 4-CP (blue lines in Fig. 9) showed three

observable phases. The first two phases are the

same as those observed for the reaction performed

in the absence of 4-CP (upper inset in Fig. 7),

indicating that these two phases represent formation

of C4a-hydroperoxy-FAD with a bimolecular rate

constant of 7.0 ± 0.2 × 10⁴M^-1.s^-1and 3.8 ± 0.3 ×

10³M^-1.s^-1, respectively, as previously explained in

Section B.1. The last phase showed a decrease of

absorbance at 380 nm and a large increase of

absorbance at 450 nm that is independent of oxygen

concentration. The kinetics of this phase are

8

Kinetic Mechanism of HadA monooxygenase

second phase (0.2-20 s) of the reaction showed a

large increase in fluorescence at Ex380, but only a

small change in fluorescence at Ex450. The

observed rate constant of this phase was

independent of oxygen concentration. This phase

likely represents formation of C4a-hydroxy-FAD

which is known to be more fluorescent than C4a-

hydroperoxy-flavin (36,37,41-43). The last phase

showed a large decrease in fluorescence at Ex380

and a large increase in fluorescence at Ex450,

implying that this phase represents a dehydration

reaction to eliminate H₂O from C4a-hydroxy-FAD

to form oxidized FAD (Step 7 in Fig. 5). A control

reaction in the absence of 4-CP was also carried out,

and the result did not show any formation or decay

of Ex380 species (Fig. S12); only the increase of

fluorescence due to formation of oxidized FAD was

observed. These results indicate that the

fluorescence signal detected from Ex380 and Em

>495 in the presence of 4-CP is indeed derived from

C4a-hydroxy-FAD. Therefore, the rate constant of

the formation of fluorescence species with Ex380

(second phase of the fluorescence trace, 0.36 ± 0.02

s^-1, k_obs,OH) represents the observed rate constant of

the incorporation of –OH from C4a-hydroperoxy-

FAD to 4-CP (the hydroxylation step, Step 5 in Fig.

5).

from observing changes in flavins (C4a-

hydroperoxy-FAD,

C4a-hydroxy-FAD

and

oxidized FAD) rather than from direct observation

of the substrate 4-CP. Therefore, the rate constant

associated with hydroxylation plus dechlorination

of 4-CP was investigated using rapid-quench flow

(RQF) techniques. The RQF experiment was

carried out in the same manner as the stopped-flow

experiments, except that only oxygen at a final

concentration of 0.13 mM was used. The reactions

were carried out in 20 mM HEPES pH 7.5 with 1

mM ascorbic acid at 25^oC in the RQF machine that

was placed inside an anaerobic glove box, and were

quenched at various time points from 0-100 s by

adding an equal volume of 0.2 M HCl (final

concentration). Samples were collected, prepared,

and analyzed by HPLC/DAD to measure the

amount of HQ formation at various times (see

Experimental Procedures) and the results are

shown by the green circled line (Fig. 9).

The observed rate constant (k_obs,Pro) of

product formation was calculated using a single

exponential equation as 0.24 ± 0.01 s^-1. This value

is in the same range as the observed rate constant of

the hydroxylation step (Section E.2). As the

reaction of HadA and 4-CP does not yield 100%

product formation, the observed rate constant from

As the product yield for 4-CP

hydroxylation is not 100%, the observed

hydroxylation rate (k_obs,OH) is the combination of the

rate constants from the coupling (Step 5 in Fig. 5)

and uncoupling paths after 4-CP binding (Steps 9 in

Fig. 5) and can be described according to the kinetic

model for a bifurcation reaction (Eq. 1). The true

hydroxylation rate constant (k_OHor k₅) that

represents the hydroxylation step can be calculated

based on the observed hydroxylation constant and

product yield according to Eq. 2. The calculation

yielded a hydroxylation rate constant for 4-CP (k_OH

or k₅) of 0.18 ± 0.02 s^-1.).

RQF

experiments

(k_obs,Pro

)

also

contain

contributions from the uncoupling (H₂O₂

elimination) path similar to the explanation in E.2.

Therefore, a rate constant associated with the

product formation steps (k_Pro) was calculated by Eq.

3-4 as 0.12 ± 0.01 s^-1.

ꢀ_{ꢁꢂꢃ,ꢗꢘꢁ}= ꢀ_ꢗꢘꢁ+ ꢀ_ꢆ

%ꢇꢈꢉꢊꢋ ꢌꢍꢎꢋꢏꢐꢑ =

Eq. 3

Eq. 4

ꢒ

ꢙꢚꢛ

ꢒ

ꢕ ꢒ

ꢖ

ꢙꢚꢛ

ꢜ

=

+

Eq. 5

ꢒ

ꢝꢚꢛ

ꢓꢔ

ꢞꢟꢠ

However, the rate constant of product

formation is an observed value resulting from

hydroxylation and chloride elimination reactions

(Steps 5&6 in Fig. 5) which can be described

according to the Eq. 5. As the hydroxylation rate

constants (k_OH) can be calculated from measuring

the kinetics of C4a-hydroxy-FAD formation

(Section E.2), the rate constant associated with

chloride elimination (k_Elior k₆) was calculated as

0.36 ± 0.01 s^-1(Step 6 in Fig. 5).

ꢀ_{ꢁꢂꢃ,ꢄꢅ}= ꢀ_ꢄꢅ+ ꢀ_ꢆ

%ꢇꢈꢉꢊꢋ ꢌꢍꢎꢋꢏꢐꢑ =

Eq. 1

Eq. 2

ꢒ

ꢓꢔ

ꢒ

ꢕ ꢒ

ꢖ

ꢓꢔ

E.3. Rate constant of product formation determined

by rapid-quenched flow experiments.

Although the previous stopped-flow

spectrofluorometric experiments could measure a

rate constant associated with the hydroxylation

step, the measurements were extrapolated only

9

Kinetic Mechanism of HadA monooxygenase

F. Kinetics of the HadA:FADH^-binary complex

reaction with O₂in the presence 4-CP at various

pHs.

DISCUSSION

Our results reported herein are the first

elucidation of the kinetic mechanism of a two-

component flavin monooxygenase that can catalyze

oxidative dechlorination of various CPs. These

enzymes are prevalent in nature especially in

microbes that have potential uses for CPs

detoxification. Analysis of the products from single

and multiple turnover reactions clearly demonstrate

that HadA prefers to use 4-CP and 2-CP more than

CPs with multiple substituents. Transient kinetics

based on stopped-flow and rapid-quench flow

experiments have shown the involvement of C4a-

hydroperoxy-FAD and C4a-hydroxy-FAD in the

Reactions of HadA:FADH^-and air

saturated 4-CP were also carried out at various pHs

from 6.5-10.5 to explore if the low product

formation at pH <7.0 and >8.2 was due to the low

yield of C4a-hydroperoxy-FAD in HadA (see

Experimental Procedures). The kinetic traces

obtained at absorbance 380 nm showed that at

different pHs, the intermediates (C4a-hydroperoxy-

FAD and C4a-hydroxy-FAD) form and decay at

different rates (Fig. S13A). However, they all have

highest absorbance values (combined signals from

the two intermediates) in a similar range (within

10% variation). This is contrast to the yield of

production formation at various pHs in which the

yield variation is greater than 50% (Fig. 4). These

data suggest that the variation of product formation

at various pHs is not due to the impairment of HadA

to form C4a-hydroperoxy-FAD but likely the

protonation status of substrate or active site

residues that can affect the hydroxylation process.

oxidative dechlorination process of

a two-

component flavin-dependent dechlorinase. The

data have also elucidated the order of substrate

binding, the catalytic steps and the rate constants

involved and have identified non-productive and

productive paths of the HadA reactions.

Data from the single turnover reactions

(Table 1) have clearly elucidated the substrate

specificity of HadA. The experiments directly

measured products resulting from the first catalytic

cycle have shown that the hydroxylation efficiency

of HadA with CPs are as follows: 4-CP ~ 2-CP >

2,4-DCP > 2,4,5-TCP > 2,4,6-TCP which is

different from the previously published results of

HadA investigation using the lysate of E. coli

overexpressing the hadA gene which showed that

the multi-substituted CPs were metabolized by the

lysate more efficiently than mono-substituted CPs

(14,15). It can be envisaged that for the lysate

experiments, the overall bioconversion process can

be influenced by other enzymes in the lysate that

can further metabolize a first product. In single

turnover experiments in which the purified HadA

was used, the results can clearly state the substrate

preference of HadA because other factors affecting

the reaction efficiency such as the ability of a

reductase to generate FADH^-and efficiency of the

FADH^-transfer between the two proteins versus the

loss of FADH^-during turnover can be avoided. The

results from single turnover reactions also agree

well with the data from multiple turnover reactions

in which purified HadA was reconstituted with C₁

reductase and G-6-PD. The data indicate that HadA

prefers to use 4-CP > 2,4-DCP > 2,4,5-TCP > 2,4,6-

TCP (Fig. 2 and Table 1).

Overall reaction of HadA. The results from

sections A-E have established the kinetic

mechanism of the HadA reaction (Fig. 5). HadA

binds FADH^-as the first substrate to form a binary

complex of HadA:FADH^-that can be equilibrated

in two forms in which both forms can react with

oxygen to form HadA:C4a-hydroperoxy-FAD. 4-

CP is the third substrate to bind to the enzyme after

the reactive intermediate C4a-hydroperoxy-FAD is

formed. The fate of the ternary complex of

HadA:C4a-hydroperoxy-FAD:4-CP bifurcates into

coupling (hydroxylation plus Cl^-elimination) and

uncoupling paths of H₂O₂elimination.

In the absence of 4-CP, the HadA:FADH^-

binary complex reacts with oxygen to form

HadA:C4a-hydroperoxy-FAD

in

which

it

eliminates H₂O₂slowly (0.005 s^-1at 25^oC). We

propose that in the absence of 4-CP in vivo, the

enzyme predominantly accumulates in the

HadA:C4a-hydroperoxy-FAD form. This way,

HadA can avoid formation of the HadA:4-CP dead-

end complex and the enzyme is ready for

dechlorination/hydroxylation activity. All kinetic

and thermodynamic parameters of the individual

steps are summarized in Table 2.

10

Kinetic Mechanism of HadA monooxygenase

Our

understanding of the regio-selectivity of two-

component flavin-dependent dechlorinases.

results

have

expanded

the

The transient kinetic studies in this report

elucidated the order of substrate binding and

identified the rate-limiting step in the overall

reaction of HadA. For the productive path, HadA

binds to FADH^-as the first substrate to form a

binary complex of HadA:FADH^-that reacts with

oxygen to result in C4a-hydroperoxy-FAD. The

detection of highly fluorescent flavin species only

after 4-CP binds to the C4a-hydroperoxy-FAD

demonstrates the involvement of C4a-hydroxy-

FAD in the HadA dechlorination. C4a-hydroxy-

FAD has been shown to be highly fluorescent in

flavin-dependent enzymes (36,37,41-43). As this

step occurs before Cl^-elimination to form

hydroquinone, the data suggest that the transfer of

a terminal –OH from C4a-hydroperoxy-FAD to 4-

CP to result in C4a-hydroxy-FAD occurs before Cl^-

elimination. Based on the values of all the rate-

constants involved in the reaction of HadA (Fig. 5

and Table 2), the hydroxylation step is a major rate-

limiting step of the HadA reaction. This result

agrees with computational calculation models of

the reaction of TftD in which the hydroxylation step

was proposed to be the rate-determining step for

dechlorination (50). The involvement of C4a-flavin

adduct species in the HadA reaction makes its

mechanism unique from the mechanisms of

reductive dehalogenation performed by tyrosine

deiodinase from human (51,52) and glutathione-

dependent dehalogenase from S. chlorophenolicum

(53).

Product analysis of 2,4,5-TCP bioconversion

indicates that HadA prefers to initially catalyzes

hydroxylation in conjunction with chloride

elimination at position 4 and then later at position 2

(Fig. 3). HadA is specific to using FADH^-as a

substrate similar to TcpA and TftD (9-13). HadA

has high sequence identity to TcpA from R.

eutropha JMP134 (86.7%). Previously, analysis of

TcpA substrate utilization was mainly done with

2,4,6-TCP and 2,4,6-TCP was specified as a native

substrate for TcpA (11-13). Previous studies of

TftD from B. cepacia AC1100 (64.2% identity to

HadA) have compared substrate preference

between 2,4,5-TCP and 2,4,6-TCP. The results

indicate that TftD can utilize 2,4,5-TCP as a

substrate faster than 2,4,6-TCP (9,10). Based on

these previous and our results, it is likely that all

three enzymes, HadA, TcpA, and TftD can use

2,4,5-TCP as a substrate more effectively than

2,4,6-TCP.

The ability of HadA to form C4a-

hydroperoxy-FAD also demonstrates that the

enzyme belongs to the Class D group of flavin-

dependent monooxygenases (22) in which the

enzymes

form

a

C4a-hydroperoxyflavin

intermediate that is necessary for the oxygenation

reaction. This is similar to the other flavin-

dependent monooxygenases such as C₂from A.

baumannii (25), luciferase from Vibrio campbellii

(38), RebH from Lechevalieria aerocolonigenes

ATCC 39243 (42), ActVA from Streptomyces

coelicolor (44), Baeyer-Villiger monooxygenase

(45), N-hydroxylating hydroxylases (46,47) p-

hydroxybenzoate hydroxylase (48), and 3-

hydroxybenzoate-6-hydroxylase (49). Previous

investigations of TftD detected a species with a

high absorption around the 390 nm region during

multiple turnover reactions which was speculated to

be a C4a-flavin adduct (9). With our current

findings, it is very likely that the species previously

detected in the multiple turnovers of TftD was

either C4a-hydroperoxy-FAD or C4a-hydroxy-

HadA also has various mechanistic

properties distinct from other enzymes that make up

the Class D monooxygenases. 4-CP can bind to

HadA to form a dead-end complex of HadA:4-CP

in which its binding obstructs the productive path

of FADH^-binding (Fig. 8). This result is different

from those of the p-hydroxyphenylacetate

hydroxylases

(members

of

Class

D

monooxygenase) from A. baumanii and

Pseudomonas aeruginosa in which the substrate

HPA cannot bind to the apoenzymes (25,26,54).

However, formation of an enzyme:substrate dead-

end complex was also observed in bacterial

luciferase (a member of the Class

C

FAD. Studies of

a

single component

monooxygenases) in which an aldehyde substrate

can bind to the enzyme and prevent the binding of

FMNH^-that is required for proceeding to a

productive path (39,40). However, as the rate

constants of FADH^-binding and formation of C4a-

pentachlorophenol hydroxylase (PcpB) from S.

chlorophenolicum (18,19) also showed the

existence of C4a-flavin adducts.

11

Kinetic Mechanism of HadA monooxygenase

hydroperoxy-FAD (Fig. 5 and Table 2) are much

faster than the rate constant of H₂O₂elimination, it

is possible that under physiological conditions and

in the absence of CPs, the enzyme mainly exists in

the C4a-hydroperoxy-FAD form. This mode would

be an efficient means for avoiding formation of a

dead-end complex of HadA:4-CP such that the

enzyme can be maintained in a state ready for the

dechlorination process. When CPs are present, the

ligand can bind to the HadA:C4a-hydroperoxy-

FAD and the reaction can promptly proceed to a

hydroperoxy-FMN formation were detected when

mixing a solution of preformed PlLuxAB:FMNH^-

with O₂, while only one phase was detected in the

non-premixed reaction (56). It is not clear why

these enzymes possess this conformational change

property. The mixture of enzyme populations that

react differently with oxygen may be involved with

the control of oxygen diffusion into the active site

or the ability of reduced flavin to form the C4a-

hydroperoxyflavin intermediate. It is known that

diffusion of oxygen into a flavin-dependent

monooxygenase can influence the rate of C4a-

hydroperoxyflavin formation (57). Recently, it has

productive

dechlorination

path.

C4a-

hydroperoxyflavin is also thought to be a major

species that accumulates during the reactions of p-

hydroxyphenylacetate hydroxylase from A.

baumanii and Pseudomonas aeruginosa under

physiological conditions (25,26,54).

HadA can bind to FADH^-and exist in two

populations consisting of two different forms of

HadA:FADH^-that can react with oxygen to form

C4a-hydroperoxy-FAD with different bimolecular

rate constants. A longer incubation time resulted in

more of the species that reacts slower with oxygen.

This property does not exist in other enzymes in the

been

shown

that

formation

of

C4a-

hydroperoxyflavin in a flavin-dependent enzyme

occurs via a mechanism of proton-coupled electron

transfer (58,59). In the C₂reaction, in which the

formation of C4a-hydroperoxyflavin is almost

barrier-less with a bimolecular rate constant at 4^oC

of 1.1 × 10⁶M^-1s^-1, a His residue that is located

nearby the flavin C4a-position is necessary to

facilitate the rapid formation of the intermediate

(25,28). When we examined the X-ray structure of

apoTcpA (PDB:4G5E) (21) that is similar to HadA

and aligned it with the C₂structure (PDB:2JBT)

(60). His290 and Arg101 could be identified as

potential proton donors at the active site. It is

possible that the conformational change of HadA to

a slower reacting species may be due to the change

in the positions of these residues with respect to the

flavin C4a-position, resulting in a slower rate of

oxygen reaction. However, the conformational

change in this case does not affect the overall

turnover of HadA because the rate-limiting step of

HadA is not the oxygen reaction step (Fig. 5 and

Table 2).

Class

D

monooxygenases such as p-

hydroxyphenylacetate hydroxylases (members of

Class D monooxygenase) from A. baumanii and

Pseudomonas aeruginosa (25,26,54). However,

similar results have been observed in a few two-

component monooxygenases in other classes.

Similar properties were reported for a flavin-

dependent halogenase or RebH (Class

monooxygenase). Preincubation of RebH and

FADH^-resulted in

less active form of

F

a

RebH:FADH^-that has a lower product yield (42).

For alkanesulfonate monooxygenase or SsuD

(Class C monooxygenase), when SsuD:FADH^-was

premixed prior to reacting with oxygenated

substrate, the amount of product formation is 4-fold

lower than that produced from the mixing of free

FMNH^-and a solution of SsuD containing

oxygenated substrate (55). However, unlike in the

HadA reaction, stopped-flow experiments of RebH

and SsuD reactions in which complexes of enzyme

and reduced flavins were preformed did not reveal

Overall, the results in this report provide a

solid basic mechanistic understanding of the flavin-

dependent oxidative dechlorinases that will be

useful for future applications. The data can also be

used for comparison with future site-directed

mutagenesis studies. As HadA homologs are found

widespread in nature, an understanding of the HadA

reaction (Fig. 5 and Table 2) also contributes to the

general understanding of reactions of its homologs

identified by SSN analysis (Fig. S1) (9,10,13,33-

35). These enzymes were identified in microbes

that showed potential in bioremediation

applications.

formation

of

the

C4a-hydroperoxyflavin

intermediate (42,55). The results of the HadA

reaction are similar to the stopped-flow results of

luciferase from Photobacterium leiognathi TH1

(PlLuxAB) in which two phases of C4a-

12

Kinetic Mechanism of HadA monooxygenase

In conclusion, this study is the first report

of the kinetic mechanism of a flavin-dependent

monooxygenase (dechlorinase) HadA from R.

pickettii DTP0062. Combined approaches of

transient kinetics have elucidated the order of

substrate binding and rate constants associated with

the individual steps. The data have also revealed the

involvement of C4a-hydroperoxy-FAD and C4a-

hydroxy-FAD in the oxidative dechlorination

process. As the usage of CPs are still widespread

world-wide, understanding how nature uses flavin

and oxygen to battle these toxic chemicals provides

knowledge that may lead to future innovations for

detoxifying chemicals hazardous to human health.

containing 5 mM ethylenediaminetetraacetic acid

(EDTA), 100 µM phenylmethane sulfonyl fluoride

(PMSF) and 1 mM dithiothreitol (DTT). Cells were

disrupted by ultrasonication before being

centrifuged at 27,000 g for one hour to discard cell

debris.

Ammonium

sulfate

((NH₄)₂SO₄)

precipitation (0-20%) was used to remove the

remaining cell debris and other contaminant

proteins. The protein fraction containing the HadA

enzyme was precipitated in 20-40% (NH₄)₂SO₄.

The resulting pellet was resuspended in ~50 ml of

30 mM NaPi pH 6.5 containing 50 mM NaCl

(buffer A) and was dialyzed against 4 liters of

buffer A. Denatured proteins were removed by

centrifugation. The remaining clear solution of

proteins was loaded onto a DEAE-sepharose^TM

fast-flow (GE healthcare) anion exchange column

(volume ~200 ml) which was pre-equilibrated with

buffer A. The column was washed with 10 column

volumes of buffer A to get rid of unbound proteins.

Elution of protein was performed using a linear

gradient of 50-400 mM NaCl in 30 mM NaPi pH

6.5. The fractions were pooled and concentrated,

and the protein was exchanged into 20 mM HEPES

pH 7.5 by passing it through a Sephadex^TMG-25

(GE healthcare) column. Purified protein was

stored at -80^oC before use. All purification

processes were performed at 4^oC. The total protein

amount was determined using a Bradford assay and

the purity of HadA was assessed by SDS-PAGE

(15%) with coomassie brilliant blue staining and

compared to standard protein markers (Enzmart

Biotech, Thailand). Concentrations of purified

HadA were determined based on absorbance 280

nm using the extinction coefficient value of 74.9

mM^-1cm^-1calculated from ProtParam tool of

ExPasy (http://web.expasy.org/protparam/).

EXPERIMENTAL PROCEDURES

Construction of the hadA gene. A synthetic gene

of hadA based on the sequence GenBank:

BAA13105 (15) was synthesized by GenScript

(New Jersey, USA). The gene contains 1,554

nucleotides that encode for 517 amino acids. The

hadA synthetic gene was digested with NdeI and

BamHI restriction enzymes (Novagen) at the 5’ and

3’ ends, respectively before it was ligated with the

E. coli expression vector, pET-11a to generate the

hadA-pET-11a vector. The sequence of the hadA-

pET-11a vector was analyzed (Macrogen) to verify

its identity.

Protein expression. The hadA-pET-11a plasmid

carrying a recombinant hadA gene was transformed

into E.coli BL21 (DE3) cells. The resulting

transformant was grown on an LB agar plate

containing 50 µg/ml ampicillin at 37^oC overnight.

Auto-induction medium (61) was used to grow cells

and induce protein expression. Briefly, a single

colony of grown cells was inoculated into 100 ml

of ZYM-5052 starter medium containing 50 µg/ml

ampicillin. The starter was incubated with shaking

at 220 rpm, 37^oC overnight. Starting culture cells

(1% of v/v) were then inoculated into six flasks of

650 ml ZYP-5052 broth containing 50 µg/ml

ampicillin. The culture was incubated with shaking

at 220 rpm, 37^oC until the OD₆₀₀reached 1.0. The

temperature was then switched to 25^oC and the

cultures were continuously shaken for an additional

16 hr. Cell paste was harvested by centrifugation

and stored at -80^oC until needed.

Identification of products from single turnover

reactions. A solution of HadA (75 µM) and FAD

(25 µM) in 20 mM HEPES pH 7.5 was placed

inside an anaerobic glove box to remove oxygen.

The anaerobic solution of HadA:FAD was reduced

by adding ~5 mg/ml dithionite stock solution into

an equivalent amount (or slightly higher due to

trace amount of oxygen) of FAD to produce

HadA:FADH^-. Absorption spectra of the enzyme

solution were monitored to ensure the complete

reduction of the complex. The resulting mixture

was mixed with an equal volume of air-saturated

substrate (0.8 mM substrate and 0.13 mM oxygen)

Protein purification. The cell paste was suspended

in 50 mM sodium phosphate (NaPi) pH 7.0

13

Kinetic Mechanism of HadA monooxygenase

in tightly closed microfuge tubes. Note that all

reactions contained excess ascorbic acid (1 mM) in

order to convert the resulting quinone product after

Cl^-elimination into hydroquinone compounds that

are more stable and easier to quantitate by HPLC

analysis. All concentrations are indicated as final

concentrations after mixing. The effect of ascorbic

acid on the catalytic cycle of HadA was tested and

it was found not to interfere with the HadA reaction.

After mixing, the final concentrations of the

reagents were half of the original concentrations.

Under these conditions, FADH^-was the limiting

reagent and the reaction only proceeds for a single

turnover. Reactions were left 15 minutes at room

temperature before being quenched by adding an

equal volume of acetonitrile. The resulting white

precipitate of denatured protein was removed by

centrifugation at 13,800 g, 4^oC for 30 min.

Supernatants were diluted three-fold in 20 mM

HEPES pH 7.5 before being filtered through an

Amicon^®Ultra-0.5 centrifugal filter device with a

molecular weight cutoff 10 kDa (Millipore).

Filtrates were collected and analyzed by

HPLC (Agilent Technologies 1100 or 1260 Infinity

series) equipped with a UV-visible diode array

detector (DAD) and/or electrospray ionization mass

spectroscopic detector (ESI-MSD) (Agilent

Technologies 6120 Quadrupole LC/MS). The

stationary phase was a Nova-Pak^®(Waters) C18

reverse phase column with a 4 µm particle size and

a 3.9 x 150 mm column size. Mobile phases were

either a gradient of H₂O/methanol containing 0.1%

formic acid or a gradient of H₂O/acetonitrile

containing 0.1% formic acid. All HPLC grade

solvents were purchased from Burdick & Jackson

(B&J) scientific Co., Ltd. The HPLC was run at

ambient temperature with a flow rate of 0.5 ml/min

and a sample injection volume of 20 µl. The MSD

was set in negative mode with a scan range of m/z

from 10-500 or selected ion monitoring (SIM)

mode that was set at the specific m/z of the substrate

and expected product. Products were analyzed by

comparing their HPLC retention time and mass

with those of standard compounds.

pH effects on HadA reactions. In order to

investigate the effects of pH on HadA, single

turnover reactions of HadA using 4-CP as a

substrate were carried out at various pHs. A pH

jump method in which an enzyme solution in a

lower ionic strength buffer at pH 7.5 was mixed

with buffers with higher ionic strength at various

pHs was employed. An anaerobic mixture of HadA

and FADH^-(75 µM and 25 µM, respectively) was

prepared in 20 mM HEPES pH 7.5 as described

above in an anaerobic glove box. To the mixture of

HadA:FADH^-was added an equal volume of air

saturated 4-CP (0.8 mM and 0.13 mM oxygen) in a

three buffer system consisting of N-(2-Acetamido)-

HPLC/diode

array

detector

(DAD)

or

HPLC/DAD/mass spectroscopic detector (MSD)

(Agilent) (see below). Commercially available

substrates and products were purchased from

Sigma-Aldrich, Merck, and Tokyo Chemical

Industry Co., Ltd. (TCI). Standard curves of each

authentic compound were constructed to calculate

the amount of product formed or substrate

consumed. Coupling percentage was calculated by

comparing the product yield with the limiting

reagent, FADH^-.

Bioconversion by multiple turnover reactions.

Multiple turnover reactions of HadA were carried

out by coupling the reaction with those of glucose-

6-phosphate dehydrogenase (G-6-PD) (Sigma) and

flavin reductase (C₁from A. baumannii). Reactions

(5 ml) contained substrate (100 µM), G-6-P (1

mM), G-6-PD (0.5 unit/ml), C₁(1 µM), NAD⁺(5

µM), FAD (10 µM), HadA (10 µM) and ascorbic

acid (1 mM) in 20 mM HEPES pH 7.5. Reactions

were stopped at various time points to separate

products from proteins similar to the protocol

described above. The amount of product formed

2-aminoethanesulfonic

acid

(ACES):Tris:ethanolamine (ionic strength = 0.1 M)

at various pHs with ascorbic acid (1 mM). This

buffer system covered a pH range of 5.5-10.5 (62).

Samples were processed and analyzed for product

formation by HPLC/DAD as described above.

Transient kinetics of the HadA reaction. Single

mixing

or

double-mixing

stopped-flow

spectrophotometers models SF-61SX and rapid-

quench flow model RQF-63 from TgK Scientific

(Bradford-on-Avon, UK) were used to monitor the

kinetics of the HadA reaction. The flow path of the

instrument was made anaerobic by flushing it with

a solution of ~5 mg/ml sodium dithionite and

leaving dithionite solution in the instrument

was

analyzed

using

HPLC/DAD

or

HPLC/DAD/MSD as described below.

Product analysis. Products from single and

multiple turnover reactions were analyzed using an

14

Kinetic Mechanism of HadA monooxygenase

overnight before use. The flow unit was rinsed three

times with anaerobic buffer that was bubbled with

nitrogen gas to remove excess sodium dithionite

before performing the experiment. To generate a

solution of the HadA:FADH^-complex, an

anaerobic solution of FAD (25 µM) and HadA (75

µM) in 20 mM HEPES pH 7.5 was

stoichiometrically reduced by adding an anaerobic

solution of sodium dithionite (~5 mg/ml) in an

anaerobic glove box. The reduced binary complex

was placed inside an anaerobic glass tonometer and

was later loaded onto the stopped-flow

spectrophotometer. A solution of the HadA:FADH^-

complex was mixed with 20 mM HEPES pH 7.5

buffer containing 4-CP and various concentrations

of oxygen at 25^oC in the stopped-flow instrument.

Oxygenated buffers were prepared by bubbling the

buffer with various mixtures of oxygen and

nitrogen gases (final concentation of 0.13, 0.31,

0.61 mM oxygen) (25). Kinetic traces were

obtained by monitoring the absorbance at

wavelengths of 380 and 450 nm. Observed rate

constants of the reactions were analyzed using

ProgramA software (developed by Chiu C. J.,

Chang R., Diverno J., and Ballou D.P., at the

University of Michigan, Ann Arbor, MI, USA). The

rate constants of the oxygen dependent phases were

plotted using KaleidaGraph software (Synergy

Software) and analyzed as a bimolecular reaction

with oxygen.

Analysis of Sequence Similarity Network (SSN).

A sequence similarity network (SSN) for HadA was

carried out using the option A of Enzyme Function

Initiative (EFI)-Enzyme Similarity Tool (EFI-EST,

http://efi.igb.illinois.edu/efi-est/) and sequences

from UniProt database that had identity to HadA of

34% or higher (63,64). SSN of HadA and its

homologs were generated using an EFI alignment

score of 70 (equivalent to grouping sequences of

>27.74% in the same cluster). Networks of these

sequences were visualized using Cytoscape version

3.2.1 (65).

ACKNOWLEDGEMENTS.

This research was financially supported by The Thailand Research Fund through grant RTA5980001, a

grant from Mahidol University (to P.C.), and a Development and Promotion of Science and Technology

Talents Project (DPST) Scholarship (to P. P.). We thank Ms. Paweena Choochuay and Ms. Philaiwarong

Kamutira from Enzmart Biotech for helping with SDS-PAGE analysis of the purified HadA.

CONFLICT OF INETEREST.

The authors declare that they have no conflict of interest with the contents of this article.

AUTHOR CONTRIBUTION.

P.P. performed experiments, analyzed results and wrote manuscript. K.T. performed dechlorinase sequence

analysis. J.S. gave suggestions on data analysis. P.C. conceived the study, analyzed results and wrote the

manuscript.

15

Kinetic Mechanism of HadA monooxygenase

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65.

19

Kinetic Mechanism of HadA monooxygenase

Table 1. Percentage of substrate dechlorination resulted from single and multiple turnover reactions

Single turnover reaction

Multiple turnover reaction

Substrate

4-CP

Product*

HQ

% Coupling

% Conversion

t_1/2(min)

49 ± 4

43 ± 7^#

100

-

13

-

2-CP

CHQ

2,4-DCP

2,4,5-TCP

2,4,6-TCP

CHQ

14 ± 1

100

35

56

nd

2,5-DCHQ

2,6-DCHQ

9.0 ± 0.2

4.0 ± 0.3

72

#

*Product was characterized in the presence of ascorbic acid. Product formation from single turnover

reactions were estimated based on the depletion of substrate. A dash (-) represents reactions that were not

performed. (nd), t_1/2of 2,4,6-TCP could not be analyzed because the reaction did not finish after 600 min.

20

Kinetic Mechanism of HadA monooxygenase

Table 2. Summary of kinetic and thermodynamic parameters for the reaction of HadA:FADH^-with

4-CP.

Kinetic and thermodynamic*

Step

HadA:FADH^-with 4-CP

parameters

k₁

4.4 ± 0.5 × 10³M^-1.s^-1

2.0 ± 0.2 µM

1

2

3

*

K_D,FADH-

k₂

k_3.1

0.14 ± 0.01 s^-1

7.0 ± 0.2 × 10⁴M^-1.s^-1

3.8 ± 0.3 × 10³M^-1.s^-1

0.18 ± 0.02 s^-1

0.36 ± 0.01 s^-1

0.12 ± 0.01 s^-1

6.9 ± 0.2 × 10^-2s^-1

5.4 ± 0.2 × 10^-3s^-1

0.18 ± 0.03 s^-1

k_3.2

5

6

k₅or k_OH

k₆or k_Eli

k_Pro

5+6

7

k₇

8

k₈

9

k₉

k₁₀

25 ± 2 M^-1s^-1

k_-10

10 ± 2 × 10^-4s^-1

10

40 ± 10 µM

*

K_D,CP

29 ± 4 µM

21

Kinetic Mechanism of HadA monooxygenase

Figure 1. Reactions of HadA and chlorophenols with or without a para- substituent. For chlorophenols

with a para- substituent, HadA catalyzes hydroxylation plus Cl^-elimination to yield quinone derivatives

that can be reduced by ascorbic acid to form hydroquinone products (upper pathway). In the absence of a

para- substituent, HadA catalyzes hydroxylation alone to yield hydroquinone (lower pathway).

22

Kinetic Mechanism of HadA monooxygenase

B) 2,4-DCP

A) 4-CP

48

100

80

60

40

20

0

100

80

60

40

20

0

80

60

40

20

0

32

16

0

40

80

120

0

100

200

300

400

Time (min)

D) 2,4,6-TCP

C) 2,4,5-TCP

48

32

16

0

100

80

60

40

20

0

80

60

40

20

0

200

400

600

0

100

200

300

400

Time (min)

Figure 2. Multiple turnover reactions of HadA and chlorophenols. Plots of the amount of substrates

remaining and product generated over time for reactions of (A) 4-CP, (B) 2,4-DCP, (C) 2,4,5-TCP and (D)

2,4,6-TCP are shown over a time course of 2-10 hr. Empty circles indicate substrate remaining in the control

reaction without HadA. Filled circles indicate substrate remaining during multiple turnover reactions.

Empty diamonds indicate product amount in the control reaction without HadA. Filled diamonds indicate

product formed during multiple turnover reactions. For (D), the amount of product from 2,4,6-TCP was

unstable.

23

Kinetic Mechanism of HadA monooxygenase

Figure 3. Catalytic reaction of HadA and 2,4,5-TCP. HadA initially catalyzes hydroxylation plus Cl^-

elimination of 2,4,5-TCP at position 4, the first hydroxylation site, to generate 2,5-DCHQ as the first

product. This step is followed by hydroxylation plus Cl^-elimination at position 2 to result in 5-CHQL.

24

Kinetic Mechanism of HadA monooxygenase

16

8

0

6

7

8

9

10

11

pH

Figure 4. Optimum pH of the HadA catalytic reaction. Amount of product (HQ) generated from 4-CP

from the single turnover reaction at various pH conditions (final pH of the reactions were 6.7, 7.1, 7.5, 7.9,

8.2, 8.7, 9.1, 9.6 and 10.0).

25

Kinetic Mechanism of HadA monooxygenase

Figure 5. Kinetic mechanism of the HadA reaction with 4-chlorophenol. FAD is oxidized FAD, FADH^-

is reduced FAD, C4aOOH-FAD is C4a-hydroperoxy-FAD, and C4aOH-FAD is C4a-hydroxy-FAD.

26

Kinetic Mechanism of HadA monooxygenase

1.2

0.8

0.4

0

0.16

0.12

0.08

0.04

0

0.1

0.2

0.3

[HadA]

(mM)

free

[HadA]

0.001

0.1

10

1000

Time (s)

Figure 6. Kinetic traces for the binding of FADH^-and HadA. Solutions of FADH^-(12.5 µM) were

mixed against various concentrations of HadA (0-0.4 mM) in 20 mM HEPES pH 7.5 at 25^oC on the stopped-

flow spectrophotometer. Results are shown in solid lines. For comparison, the re-oxidation of free FADH^-

is indicated by the dashed line. The inset shows a plot of the fraction of HadA:FADH^-formation

(ΔAbs450nm/ΔAbs450nm_max) versus the free HadA concentration.

27

Kinetic Mechanism of HadA monooxygenase

0.35

[O ]

0.3

0.2

0.1

0

2

0.25

0.15

0.05

0.4

0.3

0.2

0.1

0

0.001

0.1

10

1000

Time (s)

50

40

30

20

10

0

0.2

0.4

0.6

0.8

[O₂] (mM)

300

400

500

600

Wavelength (nm)

Figure 7. C4a-hydroperoxy-FAD in the reaction of HadA with oxygen. Main Figure. Spectra of the

three flavin species involved in the catalytic cycle including the HadA:FADH^-complex (dashed line), the

HadA:C4a-hydroperoxy-FAD intermediate obtained at 10 s after the reaction started (solid line with filled

circles) and a mixture of HadA and oxidized FAD (solid line). The upper inset shows kinetic traces of the

reaction of the HadA:FADH^-complex with oxygen monitored at 380 nm and 450 nm using stopped-flow

spectrophotometry at 25^oC, pH 7.5. The lower inset shows plots of the observed rate constants of the first

phase (filled circle) and second phase (filled square) versus oxygen concentration.

28

Kinetic Mechanism of HadA monooxygenase

A)

0.35

0.25

0.15

[4-CP]

10

5

0

2

4

6

8

10 12

[4-CP] (mM)

0.001

0.01

0.1

1

10

100

Time (s)

B)

1.2

0.8

0.4

0

0.32

0.24

0.16

0.08

0

2

4

6

8

[4-CP_free] (mM)

[4-CP]

0.001

0.01

0.1

1

10

100

Time (s)

Figure 8. Reactions of a preformed HadA:4-CP complex with FADH^-. Kinetics of the reactions

resulting from mixing a solution of air saturated HadA (75 µM) that was pre-incubated with various

concentrations of 4-CP (0.1–10 mM) with an anaerobic solution of FADH^-(25 µM) in the presence of 1

mM ascorbic acid in 20 mM HEPES pH 7.5. The reactions were monitored for absorption changes at the

wavelengths 380 nm (A) and 450 nm (B). Dashed lines in both figures represent reoxidation of FADH^-in

the absence of HadA. The inset in (A) is HQ product formation at various concentrations of 4-CP with two

different mixing modes: mixing a solution of HadA:FADH^-with an aerobic 4-CP (red square) versus

mixing an aerobic solution of HadA:4-CP with an anaerobic solution of FADH^-(blue circle). The inset in

(B) is a plot between ΔA450nm/ΔA450nm_maxversus free 4-CP concentration.

29

Kinetic Mechanism of HadA monooxygenase

16

12

8

0.4

0.3

200

100

0

[O ]

2

0.2

4

0.05

0

0.01

0.1

1

10

100

Time (s)

Figure 9. Kinetics of the reaction of HadA:FADH^-with oxygen in the presence of 4-CP. Kinetic traces

obtained from single turnover reactions of a HadA:FADH^-binary complex (75 µM and 25 µM,

respectively) with oxygenated 4-CP (0.8 mM 4-CP with 0.13, 0.31, and 0.61 mM oxygen ) in 20 mM

HEPES pH 7.5 at 25^oC. Blue lines are kinetic traces detected by stopped-flow spectrophotometry at

wavelengths 380 (solid line) and 450 nm (dashed line). Red lines are fluorescence signal detected on a

stopped-flow apparatus using the excitation wavelength of 380 nm (solid line) and 450 nm (dashed line)

with emission wavelengths >495 nm. The green line is a calculated exponential curve that fits with HQ

formation at each time point analyzed by rapid-quench flow (RQF) and HPLC/DAD techniques.

30

Article Doi

Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA

DOI: 10.1074/jbc.M116.774448

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.M116.774448

Full text of DOI:10.1074/jbc.M116.774448

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