Methane Generation and Reductive Debromination of Benzylic Position by Reconstituted Myoglobin Containing Nickel Tetradehydrocorrin as a Model of Methyl-coenzyme M Reductase

Article

Methane Generation and Reductive Debromination of Benzylic

Position by Reconstituted Myoglobin Containing Nickel

Tetradehydrocorrin as a Model of Methyl-coenzyme M Reductase

Yuta Miyazaki, Koji Oohora,* and Takashi Hayashi*

Cite This: https://dx.doi.org/10.1021/acs.inorgchem.0c00901

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sı Supporting Information

ABSTRACT: Methyl-coenzyme M reductase (MCR), which

contains the nickel hydrocorphinoid cofactor F430, is responsible

for biological methane generation under anaerobic conditions via a

reaction mechanism which has not been completely elucidated. In

this work, myoglobin reconstituted with an artiﬁcial cofactor,

nickel(I) tetradehydrocorrin (Ni (TDHC)), is used as a protein-

based functional model for MCR. The reconstituted protein,

rMb(Ni (TDHC)), is found to react with methyl donors such as

methyl p-toluenesulfonate and trimethylsulfonium iodide with

methane evolution observed in aqueous media containing

dithionite. Moreover, rMb(Ni (TDHC)) is found to convert benzyl

bromide derivatives to reductively debrominated products without

homocoupling products. The reactivity increases in the order of

primary > secondary > tertiary benzylic carbons, indicating steric eﬀects on the reaction of the nickel center with the benzylic carbon

in the initial step. In addition, Hammett plots using a series of para-substituted benzyl bromides exhibit enhancement of the

reactivity with introduction of electron-withdrawing substituents, as shown by the positive slope against polar substituent constants.

These results suggest a nucleophilic S 2-type reaction of the Ni(I) species with the benzylic carbon to provide an organonickel

species as an intermediate. The reaction in D O buﬀer at pD 7.0 causes a complete isotope shift of the product by +1 mass unit,

supporting our proposal that protonation of the organonickel intermediate occurs during product formation. Although the turnover

numbers are limited due to inactivation of the cofactor by side reactions, the present ﬁndings will contribute to elucidating the

reaction mechanism of MCR-catalyzed methane generation from activated methyl sources and dehalogenation.

INTRODUCTION

also promote dechlorination of alkyl chlorides as well as

reductive dehalogenation of alkyl iodide, brominated acids, and

bromoalkanesulfonates to produce alkanes, alkanoic acids and

■

Methyl-coenzyme M reductase (MCR), which has three

diﬀerent subunits forming a heterohexameric structure

alkanesulfonates, respectively.

The results of extensive

(

αβγ) , is known to contribute to the rate-determining and

−4

theoretical and experimental studies have led to proposals of

two plausible reaction mechanisms in the methane generation

ﬁnal step for methane formation in methanogenic archea.

Furthermore, the similar types of proteins with MCR are

reported to catalyze the reverse reaction of the last step of

methanogenesis, which is proposed as a ﬁrst step of anaerobic

oxidation of methane in a consortium that includes archaea to

promote methane oxidation and microbes to couple the

methane-derived electrons with reduction of an electron

reaction including either a CH −Ni(III) intermediate or a

1−3

transient methyl radical species.

The former intermediate is

estimated to be generated by a nucleophilic attack of the Ni(I)

species on the methyl group of the thioether moiety of methyl-

coenzyme M, followed by further electron transfer and proton

transfer from coenzyme M to achieve methane generation.

This reaction mechanism is supported by the results of

crystallographic and mechanistic investigations based on

−4

acceptor such as sulfate.

The active site of MCR has an

F430 cofactor, a natural nickel porphyrinoid consisting of the

most saturated type of monoanionic tetrapyrrole framework

which is known as hydrocorphine (Figure 1a). Previous studies

have demonstrated that the redox active behavior of F430

Received: March 26, 2020

involves the Ni(I), Ni(II), and Ni(III) oxidation states. In

methane generation, MCR catalyzes the conversion of methyl-

coenzyme M (CH S−CoM) and coenzyme B (HS−CoB) to

methane and a heterodisulﬁde (CoM−S−S−CoB) using the

active Ni(I) species of F430 (Figure 1b). MCR and F430 itself

XXXX American Chemical Society

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Article

Figure 1. (a) Chemical structure of F430. (b) Methyl-coenzyme M reductase catalyzing methane formation reaction in methanogenic archaea. (c)

Chemical structures of heme and artiﬁcial cofactors. (d) Schematic representation of the reconstitution of myoglobin with artiﬁcial cofactors.

−9

reactions of the Ni(I) species in MCR

complexes

or F430 model

an important role in promoting and regulating the enzymatic

reaction, although essentially all of the model complexes have

not been considered in terms of mimicking the eﬀect of the

protein matrix. Our group has recently developed protein-

based functional models of heme, cobalamin, and F430-

depedent enzymes by conjugation of appropriate model

complexes with apo-forms of simple and robust hemoproteins

such as myoglobin and cytochrome b₅₆₂which have hydro-

phobic cavities for cofactor binding. These hemoprotein-based

models replicate the physicochemical properties and reac-

tivities of the enzymes under physiological conditions and are

0−12

with activated alkyl reagents, especially alkyl

bromides and iodides via formation of (alkyl)C−Ni(III)

species. In the latter case, the transitional methyl radial

species is believed to be generated by homolytic cleavage of the

C−S bond of methyl-coenzyme M promoted by the Ni(I)

species, and then a hydrogen atom of coenzyme B is abstracted

to generate methane gas. This mechanism is supported by

theoretical investigations and, more recently, a single

turnover reaction using the active Ni(I) species in MCR

with less reactive coenzyme analogues.

Model complexes of F430 which have been investigated

used to evaluate the eﬀects of the protein matrix in the

10,11,15

19,20

include nickel porphyrinoids,

nickel azacyclam com-

enzymatic reactions.

Recently, nickel(II) tetradehydrocor-

2,16,17

plexes,

and a cobalamin-based nickel complex. Nickel-

rin (Ni (TDHC)) (Figure 1c) was synthesized as a model of

(

II) octaethylisobacteriochlorin (Ni(OEiBC)), which has

F430 and found to have a positively shifted redox potential for

more saturated tetrapyrrole framework compared with

porphyrin, is a suitable model because Ni(OEiBC) can be

quantitatively reduced to a Ni(I) species in organic solvents

the Ni /Ni redox process. The ﬁnding that this model is

reducible with a mild reductant such as dithionite is

advantageous because native F430 and other model systems

0,11

20a

(

Figure S1 ).

The electrochemically and chemically

require strong reductants such as Na/Hg and Ti(III) citrate.

−

prepared Ni(I) species (Ni (OEiBC) ) demonstrates reactivity

similar to that of pentamethyl ester of F430 (F430M) and

promotes reactions with alkyl halides such as methyl halides

and benzyl halide derivatives to produce reductively dehalo-

genated and homocoupling compounds. Mechanistic studies

based on spectroscopic and electrochemical kinetic studies and

product analyses indicate that the initial step occurs in a

Ni(OEiBC), an aforementioned useful model of F430, has

redox potential that is 940 mV more negative for the Ni /Ni

couple than that for the corresponding redox potential of

Ni(TDHC) because of the dianionic feature of the OEiBC

ligand in contrast to monoanionic hydrocorphine and TDHC

10a,20a

ligands.

Furthermore, insertion of Ni (TDHC) into the

apo-form of myoglobin (Mb) provides reconstituted Mb

nucleophilic S 2-type mechanism to form an organometallic

(rMb(Ni (TDHC))) as a protein-based functional model of

(

alkyl)C−Ni(III) intermediate and the second step, the

MCR (Figure 1d). The functional model demonstrates

product formation step, follows the protonation in an ionic

mechanism or hydrogen abstraction in a radical-based

mechanism depending on the polarity of organic solvents.

suﬃcient reactivity for conversion of methyl iodide to methane

gas, whereas Ni (TDHC) without the protein matrix generates

10,11

20a

a negligible amount of methane gas. This ﬁnding indicates

However, the reactive (alkyl)C−Ni(III) intermediate could

not be isolated and identiﬁed and the Ni(I) species is unstable

in aqueous media. In contrast, the protein matrix of MCR plays

the importance of the protein matrix in the reaction.

Spectroelectrochemical measurements suggest that the protein

matrix may increase the reactivity of the Ni(I) species as a

Inorg. Chem. XXXX, XXX, XXX−XXX

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Article

result of axial coordination of the proximal histidine residue,

methane generated from methyl p-toluenesulfonate and

trimethylsulfonium iodide with rMb(Ni (TDHC)) were less

20a

His93, to the nickel center.

Given the fact that rMb-

Ni (TDHC)) reacts with methyl iodide to produce methane

gas as seen in MCR, further investigation of rMb-

(

than the amounts generated by previously reported model

9a,10b,21

complexes.

This could be due to steric hindrance.

(

Ni (TDHC)) using other model substrates is expected to

However, both the methane gas generation from methyl p-

toluenesulfonate and absence of ethane gas from each substrate

provide evidence that the reaction proceeds via an ionic

mechanism in the present system, so a radical-based

mechanism is ruled out. In the case of methyl-coenzyme M,

it appears diﬃcult for the Ni(I) species to activate the C−S

bond of the thioether moiety without an appropriate reaction

scaﬀold because F430M without the protein matrix and

previous model complexes fail to generate methane from

methyl-coenzyme M in spite of negative redox potentials of the

be a useful strategy for elucidating the reaction mechanism of

MCR. In the present study, we report the reactivity of

rMb(Ni (TDHC)) in aqueous solution with respect to various

methyl donors in the methane generation reaction and with

respect to benzyl bromide derivatives in the reductive

debromination reaction. Product distribution analysis and

evaluation of the substituent eﬀect of benzyl bromide

derivatives provide essential understanding to realize the

nucleophilicity of the Ni(I) species and the S 2-type reaction

9a,10b

mechanism for reactive alkyl halides.

Ni /Ni processes and high reactivities.

In native MCR,

substrates and cofactor are precisely positioned in the reaction

site which is located at the end of a funnel-shaped substrate

channel with a length of 30 Å. Crystal structure analyses have

RESULTS AND DISCUSSION

Methane Gas Generation from Methyl Donors.

Reactions of methyl donors with rMb(Ni (TDHC)) to

generate methane were carried out in potassium phosphate

■

revealed that methyl-coenzyme M predominantly binds to the

reaction site via a salt bridge and hydrogen bonding

interactions of its sulfonate moiety with amino acid residues

to direct its thioether moiety toward the front of F430.

Coenzyme B also ﬁts within the substrate channel with salt

bridges formed between its threoninephosphate moiety and

residues near the surface of the protein matrix, resulting in its

thioheptanoyl moiety being directed toward the thioether

moiety of methyl-coenzyme M and preservation of a

hydrophobic environment during the enzymatic reaction to

protect the highly reactive Ni(I) species from bulk solvent.

Furthermore, it has been proposed that binding of substrates

to appropriate positions induces a conformational change in

MCR to move the methyl-coenzyme M cofactor closer to the

buﬀer at pH 7.0 and 25 °C in the presence of dithionite under

an N atmosphere. Dithionite was employed as a reductant in

contrast to the strong reductants Na/Hg or Ti(III) citrate

which were used in investigations of previous model

0,11,15

complexes.

Generated gases were identiﬁed and

quantiﬁed by GC. Table 1 summarizes the amount of methane

Table 1. Products of the Reaction of rMb(Ni (TDHC)) with

, ,

a b c

Methyl Donors

8b,22

nickel center of F430.

Thus, the active site of MCR is

highly optimized to promote the C−S bond cleavage. Although

TON

for

rMb(Ni (TDHC)) contains a Ni(I) species in a hydrophobic

methane

(nmol)

TON for

methane

ethane

reaction site within the Mb matrix, it does not provide a

completely optimized conﬁguration to precisely arrange the

substrate and induce the proximity eﬀect observed in native

substrate

methyl

p-toluenesulfonate

(nmol) ethane

2.2 ± 0.32

0.099 ± 0.014

n.d.

MCR, causing rMb(Ni (TDHC)) to be relatively inert with

trimethylsulfonium

2.0 ± 0.36

0.087 ± 0.016

n.d.

iodide

respect to C−S bond cleavage. The amount of methane

generated from the substrates in this study is lower than that

from the previously used methyl donor, methyl iodide. This is

due to the intrinsically higher reactivity of methyl iodide

relative to those of methyl p-toluenesulfonate and trimethyl-

sulfonium iodide. The low TON values are possibly derived

methyl-coenzyme M

n.d.

36.2 ± 0.92

n.d.

methyl iodide

1.61 ± 0.041

General reaction conditions: [rMb(Ni (TDHC))] = 45 μM,

[

dithionite] = 1.0 mM, [methyl donors] = 5.0 mM in potassium

phosphate buﬀer (100 mM, pH 7.0) at 25 °C for 7 h under an N

atmosphere. Products were identiﬁed and quantiﬁed by GC. Ref 20a.

from the unfavorable deactivation/decomposition of Ni-

Reaction conditions: [rMb(Ni (TDHC))] = 45 μM, [dithionite] =

(

TDHC).

.0 mM, [methyl iodide] = 20 mM in potassium phosphate buﬀer

Reductive Debromination Reaction of Benzylic

Position with Benzyl Bromide Derivatives. The results

(

100 mM, pH 8.0) at 25 °C for 7 h under an N atmosphere.

Products were identiﬁed and quantiﬁed by GC. n.d.: not detected.

of the methane generation reaction led to the expectation that

rMb(Ni (TDHC)) would be reactive toward alkyl halides and

gas generated from methyl donors. Methane gas generation

was observed in the reaction of methyl p-toluenesulfonate (2.2

nmol) and trimethylsulfonium iodide (2.0 nmol) (Figure S2).

In contrast, control experiments using the nickel complex itself

without the protein matrix and using only dithionite exhibited

no methane generation (Tables S1 and S2). Methyl p-

toluenesulfonate is known to have poor reactivity in a

yield reductively dehalogenated compounds. Table 2 shows the

results of the reaction of excess benzyl bromide and its

derivatives in potassium phosphate buﬀer at pH 7.0 and 4 °C

under an N atmosphere in the presence of dithionite, followed

by extraction of nonvolatile compounds with an organic

solvent. The extracted compounds were analyzed by GC/MS.

It was found that benzyl bromide is converted to toluene (9.8

μM) as a reductively debrominated product under conven-

tional conditions (Tables 2 and S3). Dibenzyl, a homocoupling

product generated via a radical-based mechanism, was not

detected. The time-course plots of generated amounts of

toluene are shown in Figure 2. The initial reaction rate

10b

radical-based mechanism.

Trimethylsulfonium iodide is

the simplest sulfonium species with an activated C−S

a,10b,21

bond.

In both reactions, no other gases such as ethane

were observed. In contrast, methyl-coenzyme M does not

promote a methane generation reaction. The amounts of

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Article

a b

Table 2. Products of the Reaction of rMb(Ni (TDHC)) with Benzyl Bromide Derivatives

General reaction conditions: [rMb(Ni (TDHC))] or [rMb(Co (TDHC))] = 45 μM, [dithionite] = 1.0 mM, [benzyl bromide derivative] = 1.0

mM in potassium phosphate buﬀer (100 mM, pH 7.0) containing 1% (v/v) acetonitrile at 4 °C for 2 h under an N atmosphere. Products were

identiﬁed and quantiﬁed by GC/MS. Acetonitrile was used as a cosolvent to dissolve the hydrophobic substrates in the water media. n.d.: not

detected. Toluene from benzyl bromide, ethylbenzene from 1-phenylethyl bromide, and cumene from cumyl bromide. Dibenzyl from benzyl

bromide, 2,3-diphenylbutane from 1-phenylethyl bromide, and dicumene from cumyl bromide. The values for k were obtained as rate constant

obs

averaged in the reaction at 5 and 10 min. The constants were veriﬁed with triple independent experiments.

constant, k , for the production of toluene was determined in

obs

the reaction within 10 min (Figure S3). rMb(Ni (TDHC))

provides 9.8 μM toluene with k of 0.39 μM·min , whereas

bare Ni (TDHC) generates signiﬁcantly less toluene. This

−

obs

indicates the importance of the protein matrix in the reaction.

The protein matrix appears to cause a redox shift in the Ni /

Ni process as a result of the axial histidine coordination to the

nickel center as suggested in our previous report. Toluene

generation was found to be essentially saturated within 2 h

possibly due to a side reaction causing deactivation and/or

decomposition of rMb(Ni (TDHC)). ESI-TOF mass spectral

analysis of rMb(Ni (TDHC)) after the reaction reveals that

the protein matrix and Ni(TDHC) are modiﬁed with several

3,24

benzyl groups (Figure S4 ).

The addition of benzyl

bromide to rMb(Ni (TDHC)) aqueous solution initiates

rapid UV−vis spectral changes with the disappearance of

peaks at 501 and 598 nm and the appearance of a new peak at

557 nm (Figure 3a). The transiently formed reaction

intermediate appears to be a (benzyl)C−Ni(III) species

which is immediately transformed to the inactivated benzyl

group adduct. The existence of a similar intermediate species

Figure 2. Time-course plots of toluene generation using rMb-

(

Ni (TDHC)) (solid circles) or Ni (TDHC) (solid square).

Conditions: [rMb(Ni (TDHC))] or [Ni (TDHC)] = 45 μM,

[

dithionite] = 1.0 mM, [benzyl bromide] = 1.0 mM in potassium

phosphate buﬀer (100 mM, pH 7.0) containing 1% (v/v) acetonitrile

at 4 °C under an N atmosphere.

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Figure 3. UV−vis spectral changes of (a) rMb(Ni (TDHC)) and (b) rMb(Co (TDHC)) before (blue line) and after (red line) addition of benzyl

bromide in potassium phosphate buﬀer (100 mM, pH 7.0) containing 1% (v/v) acetonitrile and 1.0 mM dithionite at 4 °C under an N

atmosphere.

has been suggested to be included in the reaction of

enzyme (Figures 1cd). We decided that rMb(Co (TDHC))

would be a suitable catalyst to compare with rMb-

−

10,11

Ni (OEiBC) with alkyl halides.

The red proﬁle monitored

by UV−vis spectroscopy is possibly assigned as a cofactor-

benzyl adduct linked at the meso position. The modiﬁed

cofactor by the benzyl group is expected to be irreducible with

(Ni (TDHC)). The debromination reaction of benzyl bromide

using rMb(Co (TDHC)) provides both toluene and dibenzyl

in which the formation of dibenzyl indicates distinctive

evidence of the radical-based mechanism in the product

formation step (Table 2). The homocoupling product was

obtained as a minor product possibly due to the ease of

hydrogen abstraction from the protein matrix within rMb-

(Co (TDHC)), because the concentration of the radical in the

solution will generally dominate the product distribution.

The addition of benzyl bromide to a solution of rMb-

(Co (TDHC)) in phosphate buﬀer (pH 7.0) leads to

dithionite because of the undesired redox potential shift.

Furthermore, the modiﬁed cofactor may no longer be present

in the protein matrix due to the steric hindrance, although

there is no experimental information.

Other benzyl bromide derivatives, 1-phenylethyl bromide

and cumyl bromide, were also investigated as substrates for

debromination with rMb(Ni (TDHC)) (Table 2). The

reaction of 1-phenylethyl bromide aﬀorded only ethylbenzene

(

1.0 μM) as a product. The reaction rate constant for

characteristic UV−vis spectral changes with the disappearance

of a peak at 530 nm and the appearance of new peaks at 448

and 497 nm as well as shoulders at 430 and 545 nm. Indeed,

the spectrum after the reaction is assumed to indicate

formation of a (benzyl)C−Co(III) species in a protein matrix

ethylbenzene generation was determined to be 0.057 μM·

min . This rate constant is about 7-fold less than the rate

constant determined for the reaction with benzyl bromide.

Moreover, cumyl bromide is not converted by rMb-

(

detected at all. The reactivities of a series of benzyl bromide

derivatives increase in the order of primary > secondary >

tertiary benzylic carbons, indicating steric eﬀects on the

−

Ni (TDHC)), and neither cumene nor dicumene was

according to its similarity with that of the CH −Co(III)

species in which peaks at 425 and 445 nm are formed upon

addition of methyl iodide to rMb(Co (TDHC)). Taken

together, the reductive debromination of benzyl bromide using

10b,11

reaction with the nickel center in the initial step.

rMb(Co (TDHC)) appears to proceed via the radical-based

Assuming the initial reaction follows a radical-based mecha-

nism, the reaction rate constants should increase in the reverse

order because of the stability of the alkyl radical. Thus, these

results indicate that the Ni(I) species attacks the benzylic

mechanism which includes homolytic cleavage of the C−Co

bond of the organometallic (benzyl)C−Co(III) species,

resulting in the formation of the homocoupling product.

This reaction is sharply diﬀerent from the reaction observed

position in a nucleophilic S 2-type reaction in the initial step,

for rMb(Ni (TDHC)), which does not produce dibenzyl

which is consistent with the previously suggested mechanism

derivatives derived from the generation of radical species.

−

for the reaction of Ni (OEiBC) with activated alkyl reagents.

Debromination Reaction of Benzyl Bromide Using

Cobalamin Model System. The vitamin B₁₂derivative

known as cobalamin is a cobalt complex with a monoanionic

Evaluation of Substituent Eﬀect of Benzyl Bromide

Derivatives. For further evaluation of the reductive

debromination reactivity of rMb(Ni (TDHC)), reactions

with a series of para-substituted benzyl bromides with methyl-,

,25

corrin ligand which catalyzes dehalogenation reactions. The

dehalogenation reaction of alkyl halides proceeds via the

following three steps: (1) reduction of the Co(III) species to

the Co(I) species, (2) reaction of the Co(I) species with an

electrophilic halide, and (3) homolytic cleavage of the C−Co

bond to form an alkyl radical species, which provides a

reductively dehalogenated product or dimerized compound by

iodo-, bromo-, chloro-, triﬂuoro-, cyano-, and nitro-groups

were investigated. The corresponding toluene derivatives

produced by reductive debromination were quantiﬁed by GC/

MS (Figure S5). The k_o_b_svalues for generation of the toluene

derivatives are summarized in Table S4 .

Benzyl bromide derivatives with electron-withdrawing

substituents (e.g., −CN and −NO ) and electron-donating

homocoupling. In our previous work, Mb reconstituted with

cobalt tetradehydrocorrin, rMb(Co(TDHC)), was evaluated

as a protein-based functional model of a cobalamin-dependent

substituent (e.g., −CH ) were found to exhibit higher and

lower reaction rate constants, respectively, compared to that of

benzyl bromide. Plots of the log k_o_b_svalues against the values

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Inorganic Chemistry

Article

Figure 4. Plots of log k_o_b_svs σ (a), σ (b), 0.65σ + 0.09σ (c), and 0.62σ_m_b+ 0.08σ ^•(d) for the reductive debromination reaction of para-

−

•

substituted benzyl bromides (−CH , −H, −I, −Br, −Cl, −CF , −CN, and −NO ) by rMb(Ni (TDHC)).

−

10,11

of the Hammett σ substituent constant show a strong linear

reported S 2-type mechanism in Ni (OEiBC) .

Although

correlation with the ρ value of +0.83 (R = 0.93) (Figure 4a).

Employment of the resonance-responsive Hammett σ

substituent constant slightly improves the correlation

among other σ polar substituent constants to yield a ρ

value of 0.55 (R = 0.95), whereas several σ spin-delocalization

substituent constants give low linear correlations (R = 0.50 for

0.24 for σ , and 0.71

for σ_F) (Figures 4b, S6, and S7). The positive ρ values with

Hammett plots for S 2-type reactions by organometallic

compounds generally provide broken and curved relationships

due to switching of the rate-determining step between

−

oxidative addition and reductive elimination, the continuous

linear relationship obtained in the present experiments appears

to exhibit an invariant rate-determining step because of the

highly reactive (benzyl)C−Ni(III) intermediate.

•

33,34

•

σ , 0.35 for σ , 0.46 for σ ,

•

Isotope-Labeling Experiment in the Buﬀer of D O. To

−

obtain further insights into understanding the reaction

mechanism, the reaction of rMb(Ni (TDHC)) with benzyl

bromide was carried out in a solution of D O (pD 7.0 in

phosphate buﬀer) at 4 °C for 2 h under an N atmosphere.

Analysis of the extracted products by GC/MS indicates that

the peak obtained at the same retention time as that of

authentic toluene (Figure S8a ) is shifted quantitatively by +1

mass unit, indicating the formation of toluene-d (Figure S8b).

The deuteration of the reductively debrominated product

without detection of a dimerized product indicates that the

σ polar substituent constants (ρ = 0.83 and ρ = 0.55)

indicate the formation of a negatively charged transition state

at the rate-determining step and rule out a dominant

contribution of the radical-based mechanism. To take into

account both the polar eﬀect (ρ σ ) and the spin-

•

delocalization eﬀect (ρ σ ) of the substituents, we employed

a dual-parameter equation [log k_o_b_s= ρ σ + ρ σ ].

• •

37,38

Plotting the data with the Hammett σ substituent constant

and Creary’s σ_C^•spin-delocalization substituent constant in a

•

multiple linear regression provides ρ and ρ values of +0.65

and +0.09, respectively, with the a signiﬁcantly improved linear

ﬁrst step of activation of benzyl bromide proceeds via an S 2-

type reaction with attack of the Ni(I) species on the benzylic

position to transiently form the (benzyl)C−Ni(III) species

followed by the product formation step via the ionic

mechanism which includes protonation.

correlation (R = 0.97) as well as the result using Jiang’s σ

•

polar substituent constant and σ

spin-delocalization

•

substituent constant (ρ_m_b= +0.62, ρ = +0.08, R = 0.92)

•

(

Figures 4cd). The large |ρ /ρ | ratios of 7.2 (ρ /ρ ) and 7.8

ρ /ρ ) indicate that the polar eﬀect is dominant in the

reductive debromination reaction of benzyl bromide by

rMb(Ni (TDHC)). Furthermore, the positive ρ and ρ_m_b

values are similar to those obtained with the above-mentioned

plots using single polar substituent constants. These results

provide clear support for a mechanism which includes

nucleophilic attack of the Ni(I) species on the benzylic

position in the rate-determining step of the reductive

debromination reaction. This is consistent with the previously

P C

•

mb JJ

CONCLUSIONS

■

In conclusion, it is found that the reaction of rMb-

(Ni (TDHC)) with methyl p-toluenesulfonate and trimethyl-

sulfonium iodide in the presence of dithionite leads to methane

gas generation in aqueous media, although previously reported

model complexes of F430 require a strong reductant in organic

solvent. In addition, rMb(Ni (TDHC)) converts benzyl

bromide derivatives to reductively debrominated products

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The Supporting Information is available free of charge at

Article

with characteristic reactivity, which decreases with increasing

the steric hindrance at the benzylic position and does not

provide a homocoupling product. Furthermore, the heme

pocket of myoglobin plays an important role in promoting

these reactions because the products are not formed by

protein-free Ni(TDHC) under the same conditions. Hammett

plots for the reaction with para-substituted benzyl bromides

provide support for the ionic reaction mechanism, which

occurs via a negatively charged transition state. Evidence for

this proposed mechanism is also provided by the observation

that benzyl bromide is converted to toluene-d in D O under

The amount of product was determined with the calibration curve

obtained using an authentic standard. The calibration curves were

prepared including the process of the extraction of authentic

standards in a buﬀer solution by diethyl ether to solve the artifact

by distribution of the products in water and organic phases. For the

time-course experiments, the reaction was quenched by exposure to

the air, which immediately oxidized the active Ni(I) species, just

before the extraction by diethyl ether.

To obtain data for the Hammett plots, the reactions were analyzed

at 5 and 10 min after addition of para-substituted benzyl bromides.

The values of log kobs for the debromination of para-substituted

benzyl bromides were plotted against the Hammett substituent

constants as well as polar and spin-delocalization substituent

constants for para-substituents. The values for k_o_b_s, the initial rate

constant, were obtained as averaged rate constants in the reaction at 5

and 10 min.

the same conditions. These results indicate that the reaction of

the Ni(I) species with benzyl bromide proceeds via a

nucleophilic S 2-type mechanism in the ﬁrst step to form

the transient organonickel intermediate followed by proto-

nation (Figure S9). This is proposed for the productive

mechanism, and the actual reaction with the low turnover

numbers includes a signiﬁcant nonproductive pathway to form

an inactive cofactor-benzyl adduct linked at the meso position.

Even though the reaction behavior of benzyl bromide is

diﬀerent from the reaction behavior observed for native

methyl-coenzyme M which has a thioether moiety, the present

study demonstrates a model reaction toward alkyl halide

activations in an aqueous media. The future work on the

detection and characterization of intermediates in the model

will enhance our understanding of the enzymatic mechanism of

MCR toward activated alkyl halides. Furthermore, the present

ﬁnding provides important insights into understanding the

reactivity of a low-valent nickel complex supported by a

protein scaﬀold. This also contributes to the emergent topic to

create artiﬁcial metalloenzymes catalyzing unique non-natural

reactions such as carbene insertion because several abiological

cofactors are known to show the outstanding reactivities within

For isotope-labeling reactions, the reactions were carried out in

potassium phosphate buﬀer (100 mM, pD 7.0 in D O) containing 1%

(v/v) acetonitrile with incubation at 4 °C for 2 h. The pD value was

calculated according to the previously reported formula.

All the data were veriﬁed with triple independent experiments.

ASSOCIATED CONTENT

sı Supporting Information

■

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c00901.

Instruments; materials and methods; synthesis of

sodium 2-methylthioethanesufonate (methyl-coenzyme

M); synthesis of 2-bromo-2-phenylpropane (cumyl

bromide); GC and GC/MS analyses for methane

generation and reductive dehalogenation reaction;

chemical structure of Ni(OEiBC); products of the

reaction of Ni (TDHC) with methyl donors; products of

the reaction of dithionite with methyl donors; GC traces

41−44

protein matrices optimized by mutagenesis.

of the reaction of rMb(Ni (TDHC)) with methyl p-

toluenesulfonate or trimethylsulfonium iodide in the

presence of dithionite; products of the reaction of

rMb(Ni (TDHC)) with benzyl bromide under diﬀerent

pH values; time-course plots of toluene derivatives

generation by the reductive dehalogenation of benzyl

EXPERIMENTAL SECTION

■

Methane Generation Reaction. Methyl donors (methyl p-

toluenesulfonate, trimethylsulfonium iodide, and methyl-coenzyme

M) (ﬁnal concentration: 5.0 mM) were combined with a solution of

rMb(Ni (TDHC)) (ﬁnal concentration: 45 μM) and dithionite (ﬁnal

bromide and 1-phenylethyl bromide using rMb-

concentration: 1.0 mM) dissolved in potassium phosphate buﬀer (100

mM, pH 7.0) or buﬀer containing 1% acetonitrile (v/v) in the case of

methyl p-toluenesulfonate in a 2.0 mL vial capped with silicon septum

rubber. After incubation of reaction solution (500 μL in total) at 25

(

Ni (TDHC)); ESI-TOF mass spectra for protein and

Ni(TDHC) complex of rMb(Ni (TDHC)) after the

dehalogenation reaction with benzyl bromide; values of

^l^o^g^kobs for reductive dehalogenation reaction of para-

C for 7 h, an aliquot of the headspace gas (100 μL) was taken out

substituted benzyl bromides by rMb(Ni (TDHC)) and

using a Hamilton gas-tight syringe for GC analysis. Identiﬁcation of

the product was conducted by comparing its GC retention time with

that of an authentic standard. A calibration curve obtained with an

authentic standard was employed to determine the amount of

generated methane gas. The determined amount of methane was

further corrected with both of total headspace (1.5 mL) and

calculated amount of methane gas dissolved in the solution. All the

data were veriﬁed with triple independent experiments.

tabulated Hammett substituent constants for para-

substituents; time-course plots of para-substituted

toluene derivatives generation by the reductive dehalo-

genation of para-susbstituted benzyl bromides; plots of

log k_o_b_svs σ_P⁺and σ_m_bfor the reductive dehalogenation

reaction of para-substituted benzyl bromides by rMb-

•

(

Ni (TDHC)); plots of log k vs σ , σ , σ , σ , and

obs J JJ C α

Reductive Debromination Reaction of Benzylic Position.

Benzyl bromide or its derivative (1-phenylethyl bromide or cumyl

bromide) (ﬁnal concentration: 1.0 mM) was combined with a

•

^σF for the reductive dehalogenation reaction of para-

substituted benzyl bromides by rMb(Ni (TDHC));

solution of rMb(Ni (TDHC)) or rMb(Co (TDHC)) (ﬁnal concen-

tration: 45 μM) and dithionite (ﬁnal concentration: 1.0 mM)

dissolved in potassium phosphate buﬀer (100 mM, pH 7.0)

containing 1% (v/v) acetonitrile. The mixture (500 μL in total)

was incubated at 4 °C for 2 h. After the reaction, 1,3,5-

trimethoxybenzene (ﬁnal concentration: 1.0 mM) was added as an

internal standard, followed by extraction with 500 μL of diethyl ether.

The organic layer was then analyzed by GC/MS. Identiﬁcation of the

product was conducted by comparing its retention time in the

chromatogram and mass spectrum with those of authentic standards.

GC/MS traces of the reaction using benzyl bromide

and mass spectra of generated toluene; and suggested

reaction mechanisms (PDF )

AUTHOR INFORMATION

■

Corresponding Authors

Koji Oohora − Department of Applied Chemistry, Graduate

School of Engineering, Osaka University, Suita 565-0871,

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

(7) (a) Dey, M.; Kunz, R. C.; Lyons, D. M.; Ragsdale, S. W.

Article

Japan; orcid.org/0000-0003-1155-6824; Email: oohora@

chem.eng.osaka-u.ac.jp

Takashi Hayashi − Department of Applied Chemistry, Graduate

School of Engineering, Osaka University, Suita 565-0871,

Japan; orcid.org/0000-0002-2215-935X;

Email: thayashi@chem.eng.osaka-u.ac.jp

Characterization of Alkyl-Nickel Adducts Generated by Reaction of

Methyl-Coenzyme M Reductase with Brominated Acids. Biochemistry

007, 46, 11969−11978. (b) Dey, M.; Telser, J.; Kunz, R. C.; Lees, N.

S.; Ragsdale, S. W.; Hoffman, B. M. Biochemical and Spectroscopic

Studies of the Electronic Structure and Reactivity of a Methyl-Ni

Species Formed on Methyl-Coenzyme M Reductase. J. Am. Chem. Soc.

007, 129, 11030−11032. (c) Yang, N.; Reiher, M.; Wang, M.;

Author

Harmer, J.; Duin, E. C. Formation of a Nickel-Methyl Species in

Methyl-Coenzyme M Reductase, an Enzyme Catalyzing Methane

Formation. J. Am. Chem. Soc. 2007 , 129 , 11028 −11029. (d) Sarangi,

R.; Dey, M.; Ragsdale, S. W. Geometric and Electronic Structures of

Yuta Miyazaki − Department of Applied Chemistry, Graduate

School of Engineering, Osaka University, Suita 565-0871,

Japan

III

the Ni and Methyl-Ni Intermediates of Methyl-Coenzyme M

Reductase. Biochemistry 2009 , 48 , 3146 −3156. (e) Kunz, R. C.;

Horng, Y. C.; Ragsdale, S. W. Spectroscopic and Kinetic Studies of the

Reaction of Bromopropanesulfonate with Methyl-coenzyme M

Reductase. J. Biol. Chem. 2006, 281, 34663−34676.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c00901

Notes

The authors declare no competing ﬁnancial interest.

(8) (a) Ermler, U.; Grabarse, W.; Shima, S.; Goubeaud, M.; Thauer,

R. K. Crystal Structure of Methyl-Coenzyme M Reductase: The Key

Enzyme of Biological Methane Formation. Science 1997, 278, 1457−

ACKNOWLEDGMENTS

■

1462. (b) Grabarse, W.; Mahlert, F.; Shima, S.; Thauer, R. K.; Ermler,

This work was ﬁnancially supported by Grants-in-Aid for

Scientiﬁc Research provided by JSPS KAKENHI Grant

Numbers JP15H05804, JP16H06045, JP17J00008,

JP18KK0156, and JP18K19099. We appreciate support from

JST PRESTO (JPMJPR15S2). Y.M. appreciates support from

the JSPS Research Fellowship for Young Scientists.

U. Comparison of Three Methyl-coenzyme M Reductases from

Phylogenetically Distant Organisms: Unusual Amino Acid Modifica-

tion, Conservation and Adaptation. J. Mol. Biol. 2000, 303, 329−344.

Thauer, R. K.; Lamzin, V.; Ermler, U. On the Mechanism of

Biological Methane Formation: Structural Evidence for Conforma-

tional Changes in Methyl-coenzyme M Reductase upon Substrate

Binding. J. Mol. Biol. 2001, 309, 315−330. (d) Cedervall, P. E.; Dey,

M.; Li, X.; Sarangi, R.; Hedman, B.; Ragsdale, S. W.; Wilmot, C. M.

Structural Analysis of a Ni-Methyl Species in Methyl-Coenzyme M

Reductase from Methanothermobacter marburgensis . J. Am. Chem. Soc.

2011, 133, 5626−5628.

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(

•

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•

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(

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