Origin and Control of Chemoselectivity in Cytochrome c Catalyzed Carbene Transfer into Si-H and N-H bonds

Carbene Transfer into Si −H and N −H bonds

Article

Origin and Control of Chemoselectivity in Cytochrome c Catalyzed

Marc Garcia-Borras,* S. B. Jennifer Kan, Russell D. Lewis, Allison Tang, Gonzalo Jimenez-Os é s,

Frances H. Arnold,* and K. N. Houk *

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sı Supporting Information

ABSTRACT: A cytochrome c heme protein was recently

engineered to catalyze the formation of carbon−silicon bonds via

carbene insertion into Si−H bonds, a reaction that was not

previously known to be catalyzed by a protein. High chemo-

selectivity toward C−Si bond formation over competing C−N

bond formation was achieved, although this trait was not screened

for during directed evolution. Using computational and exper-

imental tools, we now establish that activity and chemoselectivity

are modulated by conformational dynamics of a protein loop that

covers the substrate access to the iron−carbene active species.

Mutagenesis of residues computationally predicted to control the

loop conformation altered the protein’s chemoselectivity from

preferred silylation to preferred amination of a substrate containing

both N−H and Si−H functionalities. We demonstrate that information on protein structure and conformational dynamics,

combined with knowledge of mechanism, leads to understanding of how non-natural and selective chemical transformations can be

introduced into the biological world.

INTRODUCTION

introduced by directed evolution. We propose that the

conformational dynamics of a single loop near the heme play

a key role in determining the activity and chemoselectivity of

the enzyme. Finally, we use our ﬁndings to guide enzyme

engineering to switch silylation−amination chemoselectivity

and demonstrate that this altered selectivity is mediated by

new and distinct conformational dynamics of the loop.

■

The Arnold lab has expanded the catalytic repertoire of

enzymes toward new-to-nature chemistries and recently

created a carbon−silicon bond-forming enzyme by directed

evolution of Rhodothermus marinus cytochrome c (Rma cyt c),

whose native function is electron transport. The laboratory-

evolved protein (Rma cyt c V75T M100D M103E variant, Rma

TDE) is capable of catalyzing carbene insertions into Si−H

bonds to form new carbon−silicon bonds (Scheme 1). Rma

cyt c has also been evolved to eﬃciently catalyze carbene

RESULTS AND DISCUSSION

^■1

. Characterization of the Reaction Mechanism of

Rma cyt c-Catalyzed Si−H Carbene Insertions. Heme

protein-catalyzed carbene insertion reactions are proposed to

occur through the formation of a reactive iron porphyrin

carbene (IPC) intermediate, followed by transfer of the

carbene to a second substrate such as an oleﬁn or an amine N−

insertions into B−H bonds in a selective manner, high-

lighting the versatility and evolvability of this protein scaﬀold

toward unnatural carbene transfer reactions. Although the

catalytic activities of these novel enzymes are established in

,3−5

previous work,

how and why this particular protein

−10

H bond (Scheme 1).

Previous studies of iron porphyrin-

scaﬀold leads to new biocatalysts that eﬃciently and selectively

catalyze these new-to-nature reactions remains unknown. Here,

we investigate the reaction mechanism and use our mechanistic

studies to help explain how certain mutations alter and

signiﬁcantly enhance enzyme reactivity and selectivity.

We employed experimental and computational tools to

investigate the mechanism of Rma TDE-catalyzed carbene Si−

H insertion (Scheme 1a); our results are similar to those

reported by Fasan and Zhang for a similar myoglobin-catalyzed

catalyzed carbene transfer reactions have suggested that three

distinct IPC electronic states could be involved in these

reactions: the closed-shell singlet (CSS), open-shell singlet

Received: February 24, 2021

Published: April 28, 2021

carbene Si−H insertion. We then investigate how the reaction

is aﬀected by the Rma TDE protein scaﬀold and mutations

2021 American Chemical Society

J. Am. Chem. Soc. 2021, 143, 7114−7123

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Scheme 1

(

a) Stereoselective carbon−silicon bond formation catalyzed by Rma cytochrome c V75T M100D M103E (Rma TDE). Rma TDE was evolved to

catalyze carbene insertions into Si−H bonds using silane 1 and ethyl 2-diazopropanoate (Me-EDA, 2) as substrates. (b) Overlay of the X-ray crystal

structure of carbene-bound Rma TDE (1.29 Å; PDB code 6CUN; in orange) and the computationally modeled (MD + QM/MM-optimized, in

gray) structure from our previous work. Iron porphyrin carbene (IPC), mutated residues, and computationally characterized bridging water

molecule are shown in stick format. (c) Schematic description of the proposed mechanism for Rma cytochrome c-catalyzed carbon−silicon bond

formation via (1) iron−carbene formation followed by (2) carbene Si−H insertion.

−13

(

OSS), and triplet (T) states.

The protein structure,

C−Si bond formation, DFT and QM/MM calculations

showed that the closed-shell singlet transition state is lowest

in energy. Intrinsic reaction coordinate (IRC) calculations

corresponding to the lowest-energy QM/MM transition state

(TDE-TS2-CSS) show that the enzymatic Si−H insertion is

concerted but highly asynchronous, consisting of partial

hydride-transfer from the silane to the carbene carbon,

followed by C−Si bond formation (Figure 1c). In contrast,

the higher in energy triplet transition state (TDE-TS2-T)

favors a radical stepwise pathway (Figures S4 −S6). These

results are consistent with those recently reported by Fasan

and Zhang on myoglobin (Mb)-catalyzed carbene insertions

into Si−H bonds, where they used truncated model DFT

calculations and radical trap experiments to conclude that the

Mb-catalyzed carbene insertion into Si−H bonds takes place

through a concerted mechanism involving a closed-shell singlet

particularly the amino acid ligand that coordinates the iron

porphyrin, has been shown to inﬂuence the relative stabilities

of various electronic states.

To characterize the mechanism of carbene insertion into Si−

H bonds catalyzed by Rma cytochrome c M100D V75T

M103E (Rma TDE), we performed density functional theory

(

DFT) calculations using a simple truncated model (imida-

zole-ligated iron porphyrin) and hybrid quantum mechanics/

molecular mechanics (QM/MM) calculations (see Supporting

Information methods, section IIb). In our previous work, MD

simulations revealed the presence of a bridging water molecule

that establishes persistent H-bond interactions with the heme

carboxylate group and the carbonyl group of the carbene once

the IPC is formed (Scheme 1b). This interaction helps

maintain the carbene in a single conformation and controls the

stereoselectivity of the carbene transfer reaction. In order to

further characterize the impact of this water molecule on the

reaction mechanism, we explicitly considered it in the QM/

MM calculations which account for the eﬀect of the protein

environment surrounding the histidine-ligated iron porphyrin.

We considered the closed-shell singlet (CSS), open-shell

singlet (OSS), and triplet (T) electronic states along the

reaction pathway (see Supporting Information methods,

section IIa ).

transition state.

Our earlier Hammett analysis showed that electron-

donating substitutions on the aryl group of the silane increase

the initial rate of the reaction, which indicates that the Si−H

insertion step is rate-determining in Rma TDE-catalyzed

reaction. To further interrogate the reaction mechanism and

rate-determining step (RDS) experimentally, the initial-rate

primary kinetic isotope eﬀect (KIE) of the Rma TDE-catalyzed

reaction was determined using dimethyl(phenyl)silane

(PhMe Si-H) and dimethyl(phenyl)silane-d (PhMe Si-D).

The ﬁrst step of the reaction (Scheme 1c) involves iron−

carbene formation from the ethyl 2-diazopropanoate substrate

These KIE experiments reﬂect the competition between

(

Me-EDA, 2) and loss of N . Both DFT and QM/MM

PhMe Si-H vs PhMe Si-D for the carbene intermediate. The

calculations showed that the closed-shell singlet transition state

TS) is lowest in energy, while the open-shell singlet TS and

triplet TS are higher in energy (Figure 1a and Figure S4a).

KIE value obtained for Rma TDE was 2.1 ± 0.2, indicating that

Si−H bond cleavage is rate-determining in this particular

biocatalytic system (see Figure S1). The experimental initial-

rate KIE for Rma TDE is comparable to that computed for

TDE-TS2-CSS (1.74, see Table S1). Similarly, a KIE value of

(

This diﬀers from earlier studies showing the open-shell singlet

is favored with a thiolate ligand. In the second reaction step,

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Figure 1. (a) Computed reaction pathway for carbene Si−H insertion reaction between PhMe SiH 1 and Me-EDA 2 catalyzed by a model

imidazole-ligated iron porphyrin (truncated DFT model). Results with three iron electronic states are given. The calculated intrinsic electronic and

−

Gibbs free energies are given in kcal·mol . Gibbs free energies do not consider the formation of substrate−substrate or enzyme−substrate reactant

complexes, thus neglecting the stabilizing interactions that overcome the unfavorable −TΔS term of a bimolecular reaction (usually about 12 kcal·

−

mol in the free energies). (b) Optimized geometries for the lowest in energy TS1-CSS and TS2-CSS obtained from truncated QM and QM/MM

models (only atoms included in the QM region are shown for clarity). (c) Intrinsic reaction coordinate (IRC) calculation for the lowest energy Si−

H carbene insertion transition state (TDE-TS2-CSS) calculated using QM/MM (atoms included in the QM region are shown in ball and stick

−

format). Relative electronic energies are given in kcal·mol . Key distances and angles are shown in Å and degrees.

.19 was recently reported for Mb(H64V,V68A)-catalyzed

In addition to the initial-rate KIE value, the end-point KIE

value was also measured for Rma TDE and determined to be

1.34 ± 0.08 (see Figure S1 ). This is similar to the end-point

KIE values reported for Rh-, Ir-, and Cu-catalyzed reactions

reaction between ethyl α-diazoacetate (EDA) and silane 1.

The larger KIE measured for the Rma TDE-catalyzed reaction

might indicate that it corresponds to a later TS on the

concerted hydride transfer and C−Si bond-forming reaction

coordinate, as compared to the Mb(H64V,V68A)-catalyzed

reaction. This is supported by the TDE-TS2-CSS QM/MM

optimized geometry, where the C−Si and C−H bonds being

formed (2.93 and 1.42 Å, respectively, Figure 1b) are shorter

(

1.29 and 1.5 for Rh, 1.6 for Ir, 1.08 for Cu), where the C−Si

bond formation step is believed to be both rate-limiting and

concerted.

14−16

Overall, our experimental results suggest that

C−Si bond formation step is rate-limiting in the Rma TDE-

catalyzed carbene Si−H insertion reaction, while our

computations indicate that this carbene insertion step follows

a concerted, asynchronous mechanism.

(

i.e., more product-like) than those in the optimized enzyme-

free QM model TS2-CSS structure (3.07 and 1.56 Å,

respectively, Figure 1b) and the QM model TS reported by

Fasan and Zhang (3.1 and 1.7 Å, respectively). The

2. Understanding How Directed Evolution and Silane

Binding Aﬀect the Catalytic Eﬃciency of Rma TDE-

Catalyzed C−Si Bond Formation. Mutations V75T,

M100D, and M103E, introduced to wild-type Rma cyt c

during direction evolution, improve the total turnover number

occurrence of a TS in Rma TDE in which the hydride is

more transferred to the carbene is attributed to the presence of

the water molecule that is H-bonded to the carbene carbonyl

group. This hydrogen bond interaction, characteristic of the

Rma TDE enzyme, increases the electron withdrawing

character of the ester group conjugated to the carbene C

atom, which in turn enhances the electrophilicity of the

carbene. Consequently, the hydride transfer occurring during

the asynchronous but concerted process is favored (Figure 1c).

of this enzyme for C−Si bond formation. Previously, we

showed that these mutations played a role in (1) generating

space near the catalytic iron−heme cofactor and therefore

creating a new enzyme active site in Rma TDE and (2)

facilitating the access of silane substrate 1 to the active site of

carbene-bound Rma TDE by modulating the conformation

and dynamics of a loop that covers the access to the heme

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Figure 2. (a) Representative snapshots obtained from MD simulations of IPC bound in wild-type Rma cyt c (gray) and the Rma TDE variant

purple). Front loop M100/D100 and M103/E103 residues are represented in space ﬁlling format. Hydrophobic methionines (M100 and M103)

(

close the substrate channel in wild-type Rma cyt c, but mutations M100D and M103E introduce hydrophilic residues that are exposed to the

solvent in Rma TDE along the MD simulations, allowing an easier access of the silane substrate to interact with the IPC. V75T creates new key H-

bond with Y71 that induces a more hydrophobic environment for the IPC intermediate. (b) Overlay of 50 snapshots (every 10 ns) obtained from

00 ns MD simulations for wild-type Rma cyt c and Rma TDE variants in its apo and IPC-bound states. Residues mutated in the front loop

(original M100 and M103 in WT, and D100 and E103 in TDE variant) are shown in orange. Insets highlight the ﬂexibility acquired by the front

loop due to newly introduced mutations (going from “WT apo” to “TDE apo”) and the formation of the IPC (going from “WT apo” and “TDE

apo” to “WT IPC” and “TDE IPC”, respectively). Corresponding RMSF analysis are reported in Figure S8. (c) Representative snapshots of silane

binding to Rma TDE IPC in a catalytically relevant pose obtained from umbrella sampling MD simulations.

pocket (“front loop” residues 98−103) (Figure 2a,b). We

hypothesized that the protein front loop conformation and

dynamics might also modulate silane binding in a catalytically

competent pose and thus contribute to the rate-determining

step of the reaction (Scheme 1).

the silane binding to carbene-bound wild-type Rma cyt c (WT

IPC) and carbene-bound Rma TDE (TDE IPC) in a reactive

conformation (Figure S7). In both cases, the protein front loop

must change conformation to accommodate the incoming

silane substrate, and the silane substrate is only loosely bound

by both protein complexes. The silane substrate is highly

solvent exposed and not completely surrounded by the protein

Using umbrella sampling MD (US-MD) simulations (see

Supporting Information methods, section IIc ), we compared

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Figure 3. DFT optimized transition states using p-dimethylsilylaniline 6 as substrate: (a) Si−H carbene insertion; (b) N-nucleophilic attack to the

electrophilic IPC carbon atom; (c) direct N−H carbene insertion. Gibbs free energies are estimated from lowest in energy OSS IPC 5 and are given

−1

in kcal·mol . Key distances and angles are given in Å and degrees, respectively. Diﬀerent orientations of the substrate toward the carbene are

required. (d) Analysis of the substrate binding pose and near attack conformations through constrained MD simulations (N(silane)−C(carbene)

distance constrained along 500 ns MD trajectories). Representative snapshots of constrained MD simulations (constrained N−C distance to 2.5 Å,

see Supporting Information methods section IIc for details) in the wild-type and TDE IPC Rma enzyme active sites are shown. (e) C(carbene)−

N(silane)−C(silane) attack angles explored along the constrained MD simulations in wild-type (orange graph) and TDE (purple graph) IPCs. The

ideal C(carbene)−N(silane)−C(silane) attack angle value for an eﬀective N-nucleophilic attack is 121.5°, determined from DFT optimized TS.

residues (Figure 2c). Nevertheless, the silane binding aﬃnity in

the TDE IPC reactive intermediate estimated from US-MD

simulations is ∼2 kcal·mol⁻more favorable than the silane-

bound WT IPC. MD simulations show that the front loop is

more ﬂ exible in TDE IPC than in WT IPC (Figure 2b and

Figure S7 ), which allows the front loop to adopt conformations

that better favor silane binding (Figure S8 , residues 98−103).

This increased ﬂexibility in the front loop may be due to the

hydrophilicity of the D100 and E103 side chains in TDE IPC,

which are solvent exposed and are pointing outward the active

site in contrast to M100 and M103, facilitating the silane

approach to the reactive carbene (see Figure 2a,b).

From kinetics experiments, we have determined that Rma

TDE has a 7-fold increase in initial rate of carbene Si−H

insertion relative to the wild-type protein. According to

transition state theory (TST), a 7-fold increase in rate

represents an energy diﬀerence of ∼1.2 kcal·mol⁻between

the RDS barriers for wild-type Rma cyt c and Rma TDE. This

⧧

experimentally estimated energy diﬀerence (ΔΔG _R_D_S) of 1.2

−

kcal·mol agrees with the computationally estimated energy

di ﬀ erence for silane binding in a catalytically competent pose

(Figure S7). Taken together, these results describe how silane

binding in a reactive conformation diﬀers between Rma cyt c

and Rma TDE and indicate that changes in silane binding were

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Figure 4. (a) Site saturation mutagenesis of Rma TDE for chemoselective amination over Si−H carbene insertion using p-dimethylsilylaniline 6

and diazo compound 2. Reactions were performed with heat-treated cell lysates with 1 μM Rma cyt c protein, 10 mM 6, 10 mM 2, and 10 mM

sodium dithionite in M9-N buﬀer. Chemoselectivity was measured as the percent of amination product over total product formed and was

determined by gas chromatography. Total turnover number (TTN) is deﬁned as the combined concentration of 7 and 8, over protein

concentration. See Supporting Information for full description of the experimental procedures. (b) Overlay of diﬀerent snapshots obtained from

500 ns MD trajectories of Rma TDE (purple) and Rma TDFPI (gray) IPC-bound complexes. The most important structural changes correspond

to the preferred “top-open” conformation adopted by the front loop (residues 98−103) in Rma TDFPI as compared to the parent Rma TDE

variant. (c) Overlay of snapshots obtained from 500 ns MD trajectory for Rma TDFPI IPC-bound complex. The mutations N80F, M99P, and

E103I are highlighted in orange. Inset: Snapshot from constrained-MD simulation of Rma TDFPI IPC-bound complex with substrate 6, showing

how substrate 6 prefers to adopt a binding pose that favors amination via N-nucleophilic attack.

key to improving the catalytic activity of Rma cyt c toward

carbene Si−H insertions.

notably unsaturated bonds (e.g., oleﬁns and alkynes) and

26−32

nucleophiles, as well as a variety of X−H bonds.

However,

US-MD simulations also showed that the preferred

conformation of the carbene in the enzyme active site, which

such reactivity also makes chemoselectivity diﬃcult to

21,33,34

control.

Because chemoselective carbene transfer cata-

we characterized in our previous work, is not altered as the

lysts would be useful for chemical synthesis, we sought to

elucidate the principles that govern carbene transfer chemo-

selectivity. Previously, we discovered that Rma TDE diﬀer-

entiates reactions at Si−H and N−H bonds with 97%

selectivity for silylation, even though selection pressure for

chemoselectivity was not applied during the directed evolution

of this enzyme (where silane 1 was used for activity

silane approaches the enzyme. This observation, together with

the concerted nature of the Si−H carbene insertion TS we

characterized, supports our previous hypothesis of how the

cytochrome c protein controls stereoselectivity: the stereo-

chemical outcome of C−Si bond formation is solely

determined by how the silane substrate approaches the IPC

intermediate in the enzyme active site (Figures 2a and S7).

screening). This preference for Si−H functionalization diﬀers

. How Rma TDE Controls Chemoselectivity: C−Si vs

from the intrinsic reactivities of a theoretical protein-free

reaction (see below) and is the opposite of what is observed in

related carbene transfer competition experiments conducted

C−N Bond Formation. In addition to catalyzing the

formation of C−Si bonds, carbene transfer biocatalysts can

catalyze formation of other C−X bonds (X = C, N, O, S, and

,4,17−25

B).

In synthetic chemistry, metal carbenes are known

with a myoglobin enzyme. Interestingly, wild-type Rma cyt c

to be reactive toward a broad array of functional groups, most

functionalizes N−H and Si−H bonds with approximately equal

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during the entire simulation (see Supporting Information

Article

preference, which means that the mutations in Rma TDE

of new carbene transfer biocatalysts for chemical synthesis. In

order to further tailor these enzymes for new catalytic

applications, we sought to engineer and characterize an

enzyme variant with inverted chemoselectivity, favoring

amination over silylation.

somehow enhance chemoselectivity for Si−C bond formation.

The mechanisms of imidazole-ligated iron porphyrin-

catalyzed carbene transfers to N−H and Si−H bonds were

compared for the reaction of p-dimethylsilylaniline (Figure

a,b). N−H functionalization can occur through a concerted

We investigated the engineering of Rma TDE to favor the

amination product of p-dimethylsilylaniline (7) instead of the

competing silylation product (8), which Rma TDE prefers. We

hypothesized that this altered selectivity could be achieved by

modulating the conformational dynamics of the front loop. To

test this, we introduced selectivity-altering mutations to three

selected positions to modify the front loop conformation,

guided by MD simulations. Taking Rma TDE as parent

protein, the amino acid residues chosen for site-saturation

mutagenesis were E103, M99, and N80 (Figure 4). E103 (on

the front loop) was selected because it was previously shown to

N−H insertion mechanism akin to that discussed earlier for

Si−H insertion (Figure 3c) or through nucleophilic attack by

the nitrogen lone pair followed by subsequent hydrogen shift

11,19

from the ylide (Figure 3b).

By use of DFT calculations to

model the reaction between imidazole-ligated IPC and para-

dimethylsilylaniline 6, the preferred amination pathway was

determined to be N-nucleophilic attack followed by ylide

rearrangement, in agreement with Shaik’s previous mechanistic

proposal. This transition state for nucleophilic amination is

lower in energy than that for concerted Si−H insertion (ΔΔG

⧧

−

3.5 kcal·mol , Figure 3a−c); thus, in the absence of the

aﬀect the chemoselectivity. Residues M99 (on the front loop)

protein scaﬀold, amination is preferred over Si−H carbene

insertion. In addition, silylation and amination have geometri-

cally distinct transition states, as the required substrate

approaches to the carbene carbon are diﬀerent in these

reactions (Figure 3a,b).

and N80 (on the D helix of Rma cyt c) were selected because

our MD simulations suggested that these residues might play

an important role in determining front loop conformation by

establishing diﬀerent hydrogen bonding interactions between

their main and side chains (Figure S9 and Figure S10).

We generated site-saturation mutagenesis libraries at each of

the three sites and screened the variants for chemoselectivity,

as quantiﬁed by the product ratios of carbene transfer to an

amine substrate (p-isopropylaniline) and a silane substrate

(dimethylphenylsilane, 1) in a single competition reaction.

Variants displaying improved selectivity for the amination

reaction were then evaluated for their chemoselectivity on p-

dimethylsilylaniline (6). The most amination-selective variants

from each library were determined to be E103I, M99P, and

N80F, which produced the amination product as 27−34% of

the total product, as compared to 3% for the parent Rma TDE

(Figure 4a). Although only mutations at E103 had been

We investigated how chemoselectivity might be controlled

in the enzyme active site of Rma cyt c. Using carbene-bound

wild-type Rma cyt c and Rma TDE as starting structures, we

docked p-dimethylsilylaniline 6 into both enzyme active sites,

with conformation and orientation of 6 resembling how this

substrate attacks the carbene in the DFT-optimized N-

nucleophilic attack TS (Figure 3b). The docked structures

then served as starting points for MD simulations, in which the

N(substrate 6)−C(carbene) distance was kept constrained

methods, section IIc). These constrained MD simulations

showed that in wild-type Rma cyt c, 6 preferentially explores a

near attack conformation that resembles the N-nucleophilic

attack TS, in which the measured ∠C(carbene)−N(substrate

previously found to alter the enzyme chemoselectivity, our

results showed that mutations at each of these three positions

have a similar eﬀect on it. This demonstrates that mutations

that inﬂuence front loop conformational dynamics may alter

the chemoselectivity of the carbene insertion. When we

combined these three mutations in a single protein (Rma TD

N80F M99P E103I, abbreviated as Rma TDFPI), the resulting

enzyme produces the amination product as 90% of the total

product, suggesting that the front loop now occupies a new

and distinct conformation that selectively favors amination

over silylation. Rma TDFPI also exhibited ∼1.9-fold increased

turnover with respect to the parent Rma TDE variant, mainly

attributed to E103I mutation (Figure 4b).

A computational model for carbene-bound Rma TDFPI was

then generated and used to perform MD simulations to

understand the eﬀects of the new mutations. Our simulations

revealed a signiﬁcant change in front loop conformation in

Rma TDFPI compared to Rma TDE (Figure 4b). Notably, the

M99P and E103I mutations work synergistically to disfavor the

silylation transition state by changing the conformational

preference of the front loop (M99P) and blocking the

preferred binding pose required for the silane to approach

the IPC (E103I) (Figure 4c). The new active site found in

Rma TDFPI has a substrate access tunnel between the D helix

and the protein front loop that guides substrate 6 to approach

the carbene-bound heme from the top, a trajectory that favors

a near attack, amination conformation via an N-nucleophilic

attack transition state (Figure 4c). This trajectory is also

favored by the N80F mutation: due to steric repulsion between

)−C(substrate 6) angle is 121.5° (Figure 3d,e). This is in

contrast to the Rma TDE system, where 6 prefers a binding

pose that resembles the near attack conformation for the

energetically disfavored DFT optimized N−H hydrogen

⧧

−1

abstraction TS (ΔG = 41.7 kcal·mol , Figure 3c) with an

enlarged ∠C(carbene)−N(substrate 6)−C(substrate 6) angle

(Figure 3e) rather than the corresponding N-nucleophilic

attack TS. This change in the preferred near attack

conformation of the substrate is due to the new conformation

explored by the protein front loop in Rma TDE, which is

induced by mutations as previously described (Figure 3b and

Figure 2a). Taken together, DFT calculations and MD

simulations suggest that Rma TDE prevents amination by

disfavoring the N-nucleophilic attack transition state through

front loop conformation and dynamics that, at the same time,

favor silylation.

. From Mechanistic Understanding to New Protein

Function: Switching the Chemoselectivity of Rma TDE

from Silylation to Amination. The development of novel

carbene transfer enzymes has largely been driven by

repurposing existing heme proteins and evolving these proteins

,3,4,17−21,23,24,35

for improved activity for a speciﬁc reaction.

Fundamentally, the process of repurposing and evolving

enzymes for novel reactivity is an alteration of an enzyme’s

selectivity, taking advantage of their intrinsic catalytic

promiscuity. Understanding the factors that determine

enzyme selectivity may therefore facilitate the development

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Química, Universitat de Girona, 17003 Girona, Spain;

Article

the phenylalanine side chain and the protein front loop, the

front loop is now distanced from the D helix where N80F

resides. Finally, by docking substrate 6 into carbene-bound

Rma TDFPI with an amination-like conformation and

constraining the N(substrate 6)−C(carbene) distance (2.5

Å), constrained MD simulations were used to analyze the

catalytically competent binding poses of substrate 6 in Rma

TDFPI. These simulations showed that the binding pose of

substrate 6 is modulated by the new loop conformation and

hydrophobic interactions between E103I and the aromatic ring

of 6, which stabilizes the substrate in a near attack

conformation that leads to the N-nucleophilic attack TS

Detailed description of experimental and computational

methods and procedures, spectroscopic data for all new

compounds, supplementary ﬁgures and tables, and

Cartesian coordinates of DFT optimized structures

(PDF )

AUTHOR INFORMATION

■

Corresponding Authors

Marc Garcia-Borra

s − Department of Chemistry and

Biochemistry, University of California, Los Angeles,

California 90095, United States; Institut de Química

Computacional i Catalisi (IQCC) and Departament de

(Figure 4c and Figure S11). These results demonstrate that the

orcid.org/0000-0001-9458-1114; Email: marcgbq@

gmail.com

conformational dynamics of the front loop play a fundamental

role in determining the reactivity proﬁle of Rma cyt c-derived

carbene transfer catalysts and that these dynamics are easily

altered through mutagenesis, generating enzymes with altered

selectivity.

Frances H. Arnold − Division of Biology and Bioengineering

and Division of Chemistry and Chemical Engineering 210-41,

California Institute of Technology, Pasadena, California

1125, United States; orcid.org/0000-0002-4027-364X;

CONCLUSIONS

■

Email: frances@cheme.caltech.edu

The catalytic versatility and selectivity observed in engineered

carbene transfer enzymes have led to growing interest in

developing these systems for chemical catalysis. Understanding

the catalytic mechanism of these engineered enzymes and how

the protein scaﬀold inﬂuences reaction outcomes oﬀers

fundamental insights into how non-natural enzymes function

and how we might design new ones. Here, we have

characterized, through computation and experiment, how

laboratory-evolved Rma cyt c variants catalyze the formation

of carbon−silicon bonds. We determined that Rma TDE

catalyzes carbene insertion into Si−H bonds through a

concerted, nonradical mechanism and that Si−C bond

formation corresponds to the rate-limiting step. We also

found that speciﬁc H-bond interactions in the Rma TDE active

site increase the electrophilicity of the carbene as compared to

other enzymatic systems. Binding organosilane to the enzyme

in a catalytically competent pose requires unfavorable displace-

ment of the protein front loop, rendering C−Si bond

formation the rate-determining step of the overall reaction.

Directed evolution transformed Rma TDE into a faster C−Si

bond-forming enzyme by making silane binding in a catalytic

conformation more favorable in Rma TDE than in the wild-

type protein; this in turn reduces the rate-determining energy

barrier of carbene Si−H insertion. The mutations in Rma TDE

also confer chemoselectivity to the enzyme by stabilizing a

substrate binding pose that favors silylation over amination,

through modulation of the protein front loop conformational

dynamics. Finally, taking advantage of the mechanistic details

uncovered in this study and guided by computational

modeling, we introduced mutations that ultimately switched

the enzyme chemoselectivity from silylation to amination by

favoring a new conformation of the protein front loop. These

results demonstrate how protein conformational dynamics and

knowledge of reaction mechanism can guide the evolution of

new enzymatic reactions, oﬀering a framework for future

mechanism-guided engineering of useful and novel carbene

transfer enzymes.

K. N. Houk − Department of Chemistry and Biochemistry,

University of California, Los Angeles, California 90095,

United States; orcid.org/0000-0002-8387-5261;

Email: houk@chem.ucla.edu

Authors

S. B. Jennifer Kan − Division of Chemistry and Chemical

Engineering 210-41, California Institute of Technology,

Pasadena, California 91125, United States; orcid.org/

000-0001-6371-8042

Russell D. Lewis − Division of Biology and Bioengineering,

California Institute of Technology, Pasadena, California

91125, United States; orcid.org/0000-0002-5776-7347

Allison Tang − Division of Chemistry and Chemical

Engineering 210-41, California Institute of Technology,

Pasadena, California 91125, United States

Gonzalo Jimenez-Os é s − CIC bioGUNE, 48170 Derio,

Spain; orcid.org/0000-0003-0105-4337

Complete contact information is available at:

https://pubs.acs.org/10.1021/jacs.1c02146

Author Contributions

M.G.-B., S.B.J.K., and R.D.L. contributed equally. The

manuscript was written through contributions of all authors.

All authors have given approval to the ﬁnal version of the

manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health,

National Institute for General Medical Sciences Grant GM-

24480 (to K.N.H.); the National Science Foundation

Division of Molecular and Cellular Biosciences Grant MCB-

016137 and the Division of Chemical, Bioengineering,

Environmental, and Transport Systems Grant CBET-

937902 (to F.H.A.); the Spanish Ministry of Science and

■

Innovation MICINN (Grant PID2019-111300GA-I00 to

M.G.-B and Grants CTQ2015-70524-R and RYC-2013-

14706 to G.J.-O.). M.G.-B. thanks the Ramón Areces

Foundation for a postdoctoral fellowship, the Spanish

MINECO for a Juan de la CiervaIncorporación fellowship

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/jacs.1c02146.

■

121

J. Am. Chem. Soc. 2021, 143, 7114−7123

Journal of the American Chemical Society

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