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w
w
.
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a
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.
o
r
g
/
c
g
i
/
d
o
i
/
1
0
.
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0
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3
/
p
n
a
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!
1
!
Supporting!Information!for
!
Catalytic(Iron
-
Carbene(Intermediate(Revealed(in(a(Cytochrome(
c"
Carbene(Transferase
"
!
Russell!D.!Lewis
a,
1
,!Marc!Garcia
<
Borr
às
b,
1
,!Matthew!J.!Chalkley
c
,!Andrew!R.!Buller
c,
d
,
!
K
.
!
N.!
Houk
b,
2
,!S.!B.!Jennifer!Kan
c,
2
,!Frances!H.!Arnold
a,c
,
2
!
a
!
Division!of!Biology!and!Bioengineering,!California!Institute!of!Technology,!Pasadena,!CA,!91125,!USA.
!
b
!
Department!of!Chemistry!and!Biochemistry,!University!of!California!Los!Angeles,!Los!Angeles,!CA,!
90095,!USA.
!
c
!
Division!of!Chemistry!and!
Chemical!Engineering,!California!Institute!of!Technology,!Pasadena,!CA,!
91125,!USA.
!
d!
Current!address:!Department!of!Chemistry,!University!of!Wisconsin
<
Madison,!Madison,!WI,!53706,!USA
!
!
1!
Denotes!equal!author!contribution.
!
2!
To!whom!correspondence!should!b
e!addressed:!
houk@chem.ucla.edu
,!
sbjkan@caltech.edu
,!
frances@cheme.caltech.edu
!
!
!
I.
!
!
Materials!and!Methods!
!
II.
!
General!Procedures!
!
a.
!
Plasmid!construction
!
b.
!
Cytochrome!c!expression!and!purification
!
c.
!
57
Fe
<
label
l
ed!cytochrome!c!expression!and!purification
!
d.
!
Hemochrome!assay
!
e.
!
Biocatalytic!reactions
!
f.
!
Synthesis!and!
characterization
!
of!authentic!organosilicon!product
!
g.
!
UV
<
V
is!
spectroscopy
!
h.
!
X
<
ray!crystallography
!
i.
!
Mössbauer!spectroscopy
!
j.
!
Electron!paramagnetic!resonance!spectroscopy
!
k.
!
Quantum!Mechanics!(Density!Functional!Theory)
!
calculations
!
l.
!
Hybrid!
Quantum!Mechanics/Molecular!Mechanics!(QM/MM)!calculations
!
m.
!
Computational!model
l
ing!of!
Mössbauer!spectroscopic!parameters
!
n.
!
Molecular!Dynamics!(MD)!simulations
!
III.
!
Tables!and!Figures
!
IV.
!
Supplemental!References!
!
!
(
!
2
!
I.(Materials(and(Methods(
(
Unless!otherwise!noted,!all!chemicals!and!reagents!for!chemical!reactions!were!obtained!from!
commercial!
suppliers! (Acros,! Arch! Biosci
ence,! Fisher! Scientific,! Sigma
<
Aldrich,! TCI! America
,!
VWR
)!and!used!without!further!purification.!
Multitron!shakers!(Infors,!Annapolis!Junction,!MD)!
were! used! for! cell! growth.! UV
<
V
is! spectroscopy! was! performed! using! a! Shimadzu!
UV
<
1800!
spectrophotometer!(
Shimadzu,!Carlsbad,!CA).!
Sonication!was!performed!using!a!Qsonica!Q500!
sonicator.!
Silica!gel!chromatography!purifications!were!carried!out!using!AMD!Silica!Gel!60,!230
<
400!mesh.!
1
H!NMR!spectra!were!recorded!on!a!Bruker!Prodigy!40
0!MHz!instrument!and!are!
internally!referenced!to!the!residual!solvent!peak!(chloroform).!Data!for!
1
H!NMR!are!reported!in!
the!conventional!form:!chemical!shift!(δ!ppm),!multiplicity!(s!=!singlet,!d!=!doublet,!t!=!triplet,!q!=!
quartet,!hept!=!heptet,!m!=!mu
ltiplet,!br!=!broad,!app!=!appears!as),!coupling!constant!(Hz),!
integration.!Gas!chromatography!(GC)!analyses!were!carried!out!using!a!Shimadzu!GC
<
17A!gas!
chromatograph,!a!
flame!ionization!detector!(
FID
)
,!and!J&W!HP
<
5!(30!m!x!0.32!mm,!0.25!μm!filmk!
90!°C!h
old!1!min,!90!to!110!°C!at!15!°C/min,!110!to!280!°C!at!60!°C/min,!280!°C!hold!1!min,!6.2!
min!total).!Analytical!chiral!high!pressure!liquid!chromatography!(HPLC)!was!performed!with!a!
HP
<
Agilent! 1100! series! instrument! using!
i
<
PrOH! and! hexanes
!
as! the! mobile!
phase,! with!
visualization!at!210!nm.!A!Chiralcel!OD
<
H!column!was!used!for!chiral!separations!(4.6!mm!x!25!
cm).!
Plasmid!pET22!was!used!as!a!cloning!vector,!and!cloning!was!perf
ormed!using!Gibson!assembly
!
(
1
)
.!The!cytochrome!
c#
maturation!plasmid!pEC86
!
(
2
)
!
was!used!as!part!of!a!two
<
plasmid!system!
to!express!prokaryotic!cytochrome!
c#
proteins.!Cells!were!grown!using!L
ysogeny!Broth!
medium!
or! HyperBroth! (AthenaES
,! Baltimore,! MD
)! with! 100! μg/mL! ampicillin! and! 20! μg/mL!
chloramphenicol! (
LB
amp/chlor
!
or! H
B
amp/chlor
)
.! Primer! sequences! are! available! upon! request.!
Electrocompetent!
Escherichia#coli#
cells!were!prepared!following!the!protocol!of!Sambrook!
et#al
.
!
(
3
)
.
!
T5!exonuclease,!Phusion!polymerase,!and!
Taq#
ligase!were!purchased!from!New!England!
Biolabs!(NEB,!Ipswich,!MA).!
Sodium!phosphate!(NaPi,!20!mM,!pH!7.5)!
was!used!as!a!buffering!
system!for!purified!proteins,!unless!otherwise!specified.!
M9
<
N!minimal!medium!(abbreviated!as!
M9
<
N!bufferk!pH!7.4)!
was!used!wi
thout!a!carbon!sourcek!it!contains!47.7!mM!N
a
2
HP
O
4
,!22.0!mM!
K
H
2
P
O4
,!8.6!mM!NaCl,!2.0!mM!MgS
O
4
,!and!0.1!mM!CaC
l
2
.!
(
II.(General(Procedures(
(
!
(A)(Plasmid(construction.
!!!
!
!
All! variants! described! in! this! paper! were! cloned! and! expressed! using! the! pET22(b)+!
vector!
(
MilliporeSigma,!St.!Louis,!MO
).!
The!gene!encoding!
Rhodothermus#marinus
!
cyt!
c
!
V75T!M100D!
M103E!
(
Rma#
TDE)!
was!obtained!as!a!single
!
gBlock!(
Integrated!DNA!Technologies,!Coralville,!
IA
)
!
(
4
)
,! codon
<
optimized
!
for!
E.#coli
,! and! cloned! using! Gibson
!
assembly
!
(
1
)
!
into! pET22(b)+!
between!restriction!sites!
Nde
I!and!
Xho
I
!
in!frame
.!
The!gene!encoding!
Rma#
TDE
!
contained
!
an!
N
<
terminal!pelB!leader!sequence
!
and!6xHisTag
!
(
MKYLLPTAAAGLLLLAAQPAMA
HHHHHH
)
!
and!
had!the!first!seven!amino!acids!of!mature,!wild
<
type!
Rma#
cyt!
c#
removed!(
TESGTAA)
.!This!plasmid!
was!co
<
transformed!with!the!cytochrome
!
c
!
maturation!plasmid!pEC86
!
(
2
)
!
into!
E.#cloni
®
!
EXPRESS!
BL21(DE3)!cells
!
(Lucigen
,!Middleton,!WI
).
!
!
!
DNA! coding! sequence! of!
Rma#
cyt!
c#
V75T! M100D! M103E
!
with!
an!
N
<
terminal!
pelB! leader
!
sequence
!
and!6xHisTag
!
!
!
3
!
ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCG
ATGGCCCATCATCATCATCACCACCAAGACCCGGAAGCACTGGCAGCGGAAATTGGTCCG
GTCAAACAGGTGAGCCTGGGTGAACAGATTGATGCGGCCCTGGCGCAACAGGGAGAACA
GCTCTTCAACACGTATTGTACTGCGTGCCACCGTCTGGATGAGCGTTTTATCGGACCGGCC
CTGCGCGATGTTACCA
AACGTCGTGGGCCGGTTTACATCATGAACACGATGCTGAACCCG
AATGGGATGATCCAGCGTCATCCGGTGATGAAACAGCTCGTGCAGGAATATGGGACCATG
GATACCGATGAGGCCCTGAGTGAAGAACAAGCGCGCGCAATTCTGGAGTATCTGCGCCAG
GTTGCGGAAAACCAGTAATGA
!
!
Amino!acid!sequence!of!
Rma#
cyt!
c#
V75T!M100D!M103E!with!
an!
N
<
terminal!
pelB!leader
!
sequence
!
and!6xHisTag
!
!
MKYLLPTAAAGLLLLAAQPAMAHHHHHHQDPEALAAEIGPVKQVSLGEQIDAALAQQGEQLFN
TYCTACHRLDERFIGPALRDVTKRRGPVYIMNTMLNPNGMIQRHPVMKQLVQEYGTMDTDEAL
SEEQARAILEYLRQVAENQ
!
!
(B)(Cytochrome(
c
(
expression(and(purification.
!
!
!
Purified!cytochrome!
c
!
proteins!were!prepared!as!follows.!In!a!4!L!flask,!one!liter!HyperBroth!
(AthenaES)! containing! 100! μg/mL! ampicillin! and! 20! μg/mL! chloramphenicol! (HB
amp/chlor
)
!
was!
inoculated!with
!
an!overnight!culture!(20!mL,!
Lysogeny!Broth,!with!100!μg/mL!ampicillin,!20!μg
/mL!
chloramphenicol,!
LB
amp/chlor
)!of!recombinant!
E.#cloni
®
!
EXPRESS!BL21(DE3)!cells!containing!a!
pET22(b)+!plasmid!encoding!the!cytochrome!
c
!
variant,!and!the!pEC86!plasmi
d.!The!culture!was!
shaken!at!37
°C!and!200!rpm!(no!humidity!control)!until!the!OD
600
!
was
!
0.7!(approximately!3!hours).!
The!culture!was!placed!on!ice!for!30!minutes,!and!isopropyl!β
<
D
<
1
<
thiogalactopyranoside!(IPTG)!
and! 5
<
aminolevulinic! acid! (ALA)! were! added! to! final! concentrations! of! 20!
μM! and! 200! μM
,
!
respectively.
!
!
The!incubat
or!temperature!
was!reduced!to!25
°C,!and!the!culture!was!allowed!to!shake!for!20!
hours!at!200!rpm.!Cells!were!harvested!by!centrifugation!(4!°C,!15!min,!4,000xg),!and!th
e!cell!
pellet!was!stored!at!−20
°C!until!further!use!(at!least!24!hours).!The!cell!pellet!was!resuspende
d!
in!buffer!containing!100!mM!NaCl,!20!mM!imidazole,!and!20!mM!Tris
<
HCl!buffer!(pH!7.5!at!25!°C)!
and!cells!were!lysed!by!sonication!(4!minutes,!1!second!on,!1!second!off,!30%!duty!cyclek!Qsonica!
Q500!sonicator).!Cell!lysate!was!placed!in!a!75!°C!heat!bath!
for!10!minutes,!and!cell!debris!was!
removed!by!centrifugation!for!20!min!(
30,
000xg,!4!°C).!Supernatant!was!sterile!filtered!through!a!
0.45!
μ
m! cellulose! acetate! filter! and! purified! using! a! 1! mL! Ni
<
NTA! column! (HisTrap! HP,! GE!
Healthcare,! Piscataway,! NJ)! using
!
an! AKTA! purifier! FPLC! system! (GE!
H
ealthcare).! The!
cytochrome!
c
!
protein!was!eluted!from!the!column!by!running!a!gradient!from!20!to!500!mM!
imidazole!over!10!column!volumes.
!
!
The!purity!of!the!collected!cytochrome!
c
!
fractions!was!
analyzed
!
using!sodium!dodec
yl!sulfate
<
polyacrylamide!gel!electrophoresis!(SDS
<
PAGE).!Pure!fractions!were!pooled!and!concentrated!
using!a!3!kDa!molecular!weight!cut
<
off!centrifugal!filter
.!!Protein!for!crystallography!
was!
dialyzed!
overnight!into!
20!mM!Tris
<
HCl!buffer!(pH!7.5!at!25!°C
)!
using!3
.5
!
kDa!molecular!weight!cut
<
off!
dialysis!tubing.!
Protein!for!non
<
crystallography!use!was!dialyzed!overnight!into!20!mM!sodium!
phosphate!buffer!(pH!7.5)!using!3.5!kDa!molecular!weight!cut
<
off!dialysis!tubing.!
The!dialyzed!
protein!was
!
further
!
concen
trated!
using!a!3!kDa!molecular!weight!cut
<
off!centrifugal!filter
,!flash
<
froze
n!on!dry!ice,!and!stored!at!−20
°C.!
!
!
The!concentration!of!cytochrome!
c
!
was!determined!in!triplicate!using!the!
hemochrome
!
assay!
described!in!section!(
D
).
!
!
4
!
(
(C)(
57
Fe
-
label
l
ed(cytochrome(
c
(
expression(and(purification.
!
!
!
In!order!to!prepare!protein!samples
!
for!Mössbauer!spectroscopy
,!
57
Fe
<
label
l
ed!
Rma#
cyt!
c#
was!
expressed!and!purified,!using!a!protocol!
adapted!
from!Liptak
!
et#al
#
(
5
)
.!
!
!
For!1!L!
57
Fe!media
:
!
!
950!mL!
!
b
asic!salts!
buffer
!
10!mL
!
t
race!metals!mix
!
10!mL
!
v
itamin!cocktail
!
300!μL!
!
57
Fe!/!H
2
SO
4
!
solution
!
10!mL
!
1!M!MgCl
2
!
5!g
!
L/D
<
g
lucose
!
1!g
!
NH
4
Cl
!
16.8!mg
!
5
<
a
minolevulinic!acid!(ALA)
!
30!mg
!
t
hiamine!hydrochloride
!
1!mL!
!
20!mg/mL!chloramphenicol!in!ethanol
!
1!mL!
!
100!
mg/mL!ampicillin!in!water
!
!
!
For!950!mL!
b
asic!
s
alts!
b
uffer:
!
•
!
950!mL!
w
ater
!
•
!
13.0!g!KH
2
PO
4
!
•
!
13.1!g!K
2
HPO
4!
·(3H
2
O)
!
•
!
17.1!g!Na
2
HPO
4!
·(7H
2
O)
!
•
!
3.6!g!Na
2
SO
4
!
!
Adjusted#pH#to#7.4#with#5M#NaOH,#
autoclaved,#and#stored#at#room#temperature.
#
!
For!100!mL!Trace!metals!mix:
!
!
•
!
90!mL!
w
ater
!
•
!
600!mg!
c
alcium!chloride,!dihydrate
!
•
!
120!mg!
m
anganese!chloride,!tetrahydrate
!
•
!
80!mg!
c
obalt!chloride,!hexahydrate
!
•
!
70!mg!
z
inc!sulfate,!heptahydrate
!
•
!
30!mg!
c
opper!chloride,!dihydrate
!
•
!
2!mg!
b
oric!acid
!
•
!
500!mg!EDTA
!
#
Adjusted#pH#to#7.4#with#5M#NaOH,
#
and
#
ad
ded#water#until#final#volume#was#100#mL.##
Solution#was#deep#purple.
#
The#solution#was#s
terile#filte
red#through#a#0.2#μm
#
PES#
membrane#and#stored#at#
!
20°C.
#
!
For!100!mL!
v
itamin!
c
ocktail
:
!
!
•
!
100!mL!
w
ater
!
•
!
10!mg!
b
iotin
!
•
!
10!mg!
c
holine!chloride
!
!
5
!
•
!
10!mg!
f
olic!acid
!
•
!
10!mg!
n
icotinamide
!
•
!
10!mg!
c
alcium!D
<
pantothenate!
!
•
!
10!mg!
p
yridoxal!phosphate
!
•
!
1!mg!
r
iboflavin
!
!
The#solution#was#sterile#filtered#through#a#0.2#μm#PES#membrane,#and#stored#at#
!
20°C.
#
!
For!600
!
μ
L!
57
Fe
!
/
!
H
2
SO
4
!
solution
:
!
!
•
!
25.0!mg!
57
Fe!(95%!enrichment,!Isoflex!USA,!San!
Fran
c
isco)
!
•
!
600!μL!2!M!H
2
SO
4
!
!
H
2
SO
4
#
was#added#to#the#iron#powder#under#anaerobic#conditions,#and#allowed#to#shake#
overnight,#until#the#iron#was#dissolved.#The#solution#was#pale#blue,#and#was#stored#at#
room#temperature#under#anaerobic#conditions.#
#
!
!
Purified!
57
Fe!
cytochrome!
c
!
proteins! were! prepared! as! follows.!
In! a! 125! mL! flask,! 30! mL! of!
HB
amp/
chlor
!
was
!
inoculated!with!
an!overnight!culture!(1!mL,!LB
amp/chlor
)!of!recombinant!
E.#cloni
®
!
EXPRESS!BL21(DE3)!cells!containing!a!pET22(b)+!plasmid!encoding!the!
cytochrome!
c
!
variant,!
and!the!pEC86!plasmi
d.!The!culture!was!shaken!at!37
°C!and!200!rpm!(no!humidity!control)!until!
the!OD
600
!
was!0.7!(approximately!3!hours).!
In!a!4!L!flask,!one!liter!
of!
57
Fe!medi
um
!
was!inoculated!
with
!
20!mL
!
of!the!late
<
log!phase!HB
!
amp/chlor
!
culture
.!The!culture!was!
again!shaken!at!37
°C!and!
200!rpm!(no!humidity!control)!until!the!OD
600
!
was!0.3!(approximately!4!more
!
hours).!The!culture!
was! placed! on! ice! for! 30! minutes,! and! isopropyl! β
<
D
<
1
<
thiogalactopyranoside! (IPTG)! and! 5
<
aminolevul
inic!acid!(ALA)!were!added!to!final!concentrations!of!20!
μM!and!200!μM
,
!
respectively.
!
!
After!addition!of!IPTG!and!ALA,!the!culture!was!returned!to!the!shaker
,!allowed!to!shake!for!2
4
!
hours!at
!
37
!
°C,!200!rpm.!Cells!were!harvested!and!lysed!using!the!same!procedure!as!described!
in!(B).!
57
Fe
<
label
l
ed!protein!was!then!purified!as!described!in!(B).
!
!
(D)(Hemochrome(assay.
!
!
!
A!solution!of!sodium!dithionite!(10!mg/mL)!was!prepared!in!M9
<
N!buffer.!Separate
ly,!a!solution!of!
1!M!NaOH!(0.4!mL)!was!mixed!with!pyridine!(1!mL),!followed!by!centrifugation!(10,000xg,!30!
seconds)!to!separate!the!excess!aqueous!layer!gave!a!pyridine
<
NaOH!solution.!To!a!cuvette!
containing!
1
0!μL!
purified!
protein!solution
!
and
!
690!μL
!
M9
<
N!buffer,!50!μL!of!dithionite!solution!and!
250!μL!pyridine
<
NaOH!solution!were!added.!The!cuvette!was!sealed!with!Parafilm,!and!the!UV
<
Vis!spectrum!was!recorded!immediately.!Cytochrome!
c
!
concentration!was!determined!using!ε
550
<
535
!
=!22.1!mM
<
1
cm
<
1
!
(
4
,
6
).
!
!
(E)(Synthesis(and(
characterization
(
of(authentic(organosilicon(product
!
!
Ethyl!2
<
(dimethylphenylsilyl)propanoate!was!prepared!in!a!similar!
manner!to!that!published!in!
Kan!et!al.!2016
!
(
4
)
.
(
In!a!25!mL!round!bottom!flask,!0.281!g!of!dimethylphenylsilane!(2.0!mmol,!
2.0!equiv.)!were!added,
!
followed!by,!Rh
2
(OAc)
4
!
(1.1!mg,!0.25!mol%)!and!5!mL!of!
dichloromethane.!The!mixture!was!cooled!to!
0
°C,!after!which!0.128!g!of!ethyl
<
2
<
diazopropanoate!(1.0!mmol,!1.0!equiv.)!was!added!dropwise!to!the!solution.!The!reaction!was!
allowed!to!slowly!warm!up!to!room!temperature!over!4!hours!and!stirred!at!room!temperature!
for!another!1!hour.!Evaporation!of!the
!
organic!solvent!and!purification!by!silica!column!
!
6
!
chromatography!using!EtOAc!and!hexane!as!eluents!afforded!the!organosilicon!product!
ethyl!2
<
(dimethylphenylsilyl)propanoate!
with!54%!yield.!
1
H!NMR!
was!used!to!confirm!product!identity!
and!purity.
!
!
(F)(Bioc
atalytic(reactions
!!!
!
!
In!an!anaerobic!chamber,!10!μL!of!80!μM!purified!protein!was!added!to!350!μL!of!20!mM!sodium!
phosphate!buffer!(NaPi,!pH!7.5)!in!a!2!mL!crimp!vial.!!Next,!the!protein!was!reduced!by!adding!
20!μL!Na
2
S
2
O
4
!
(200!mM!in!NaPi,!pH!7.5),!and!t
he!reaction!was!initiated!with!the!addition!of!
reactants,!10!μL!PhMe
2
SiH!(400!mM!in!acetonitrile)!and!10!μL!Me
<
EDA!(400!mM!in!MeCN).!The!
vial!was!crimp!sealed,!removed!from!the!anaerobic!chamber,!and!shaken!at!400!rpm!at!room!
temperature!for!2!hours.!At!th
e!end!of!the!reaction,!the!crimp!vial!was!opened!and!the!reaction!
was! quenched! with! cyclohexane! (1! mL).! Internal! standard! was! added! (20! μL! of! 1,2,3
<
trimethoxybenzene!in!toluene)!and!the!reaction!mixture!was!transferred!to!a!microcentrifuge!tube,!
vortexed!(
1!minute),!then!centrifuged!(14,000xg,!5!min)!to!completely!separate!the!organic!and!
aqueous!layers.!The!organic!layer!(750!μL)!was!removed!for!GC!and!chiral!HPLC!analysis.!All!
biocatalytic!reactions!were!performed!in!triplicate.!The!total!turnover!numbers
!
(TTNs)!reported!
are!calculated!with!respect!to!the!protein!catalyst!and!represent!the!total!number!of!turnovers!
obtain
ed
!
using!
the!catalyst!under!the!stated!reaction!conditions.
!
!
Concentration!of!organosilicon!product!was!calculated!based!o
n
!
the!ratio!of!areas!between!the!
product!peak!and!internal!standard!peak
,!as!measured!by!GC
<
FID.
!
!
Stock!s
olutions!of!chemically!
synthesized
!
ethyl!2
<
(dimethylphenylsilyl)propanoate
!
were!prepared!
at!various!concentrations!(20!to!200!mM!in!MeCN).!To!a!microcen
trifuge!tube!were!added!360!
μL!20!mM!sodium!phosphate!buffer,!20!μL!Na
2
S
2
O
4
!
(200!mM!in!NaPi,!pH!7.5),!20!μL!organosilicon!
product,! 20! μL! internal! standard! (20! mM! 1,2,3
<
trimethoxybenzene! in! toluene)! and! 1! mL!
cyclohexane.! The! mixture! was! vortexed! (1! minute)!
then! centrifuged! (14,000xg,! 5! min)! to!
completely!separate!the!organic!and!aqueous!layers.!The!organic!layer!(750!μL)!was!removed!
for!GC!analysis.!Each!concentration!was!prepared!in!duplicate,!and!both!data!points!are!shown!
for!each!concentration
!
(Figure!S1
1)
.!The!standard!curves!plot!product!concentration!in!mM!(x
<
axis)!against!the!ratio!of!product!area!to!internal!standard!area!on!the!GC!(y
<
axis).!
!
!
Enantiomeric!exces
s!for!biocatalytically!
synthesized
!
products!w
as
!
determined!using!chiral!HPLC,!
with!
a!
Chiralcel!OD
<
H!column
!
as!the!stationary!phase!and!
1%!
i
sopropanol
,!
99%!
h
exanes
!
as!the!
mobile!phase
,!
at
!
40ºC
!
for
!
30!minutes.
!
(
(
G
)(UV
-
Visible(spectroscopy
!!!
!
!
UV
<
Visible!s
pectra!were!collected!at!room!temperature
!
between!600!and!380!nm!on!a!UV
<
1800!
Shimadzu!
spectrophotometer!in!quartz!cuvettes
!
sealed!with!Parafilm
!
(aerobic!conditions)!or!a!
screw!cap!with!a!pierceable!septum!(an
a
erobic!conditions)
.!Sodium!phosphate!(
NaPi,!
20!mM,!pH!
7.5)!was!used!as!a!buffer!system
.!
Spectra!of!reduced!
Rma#
TDE
!
was!taken!in!buffe
r!supplemented!
with!50!mM!sodium!dithionite!to!ensure!complete!reduction!of!the!
ferrous!iron
,!and!various!co
<
solvents! were! added! to! demonstrate! that! the! spectral!
characteristics
!
are! similar! in! 1%!
isopropanol,!5%!acetonitrile,!and!15%!dimethylsulfoxide
.
!
Spe
ctra!of!reduced,!Me
<
EDA
<
treated!
Rma#
TDE! was! taken! in! buffer! containing! 50! mM! sodium! dithionite,! 10! mM! ethyl! 2
<
diazo!
propanoate,!
and!1%!isopropanol.!
Co
<
solvents!were!added!to!demonstrate!that!the!addition!of!5%!
acetonitrile!or!15%!dimethylsulfoxide!did!not!
substantially!impact!the!spectrum.!!
For!spectra!taken!
under! anaerobic! conditions,! the! headspace! of! the! cuvettes! was! de
<
gassed! with! argon! for! 5!
minutes!prior!to!the!addition!of!sodium!dithionite!and!Me
<
EDA.!!
!
!
7
!
!
(
H
)(X
-
ray(crystallography
!!!
!
!
Fine!screening!for!
Rma#
TDE!
crystallization! was! performed! around! crystallography! conditions
!
previously!reported!to!yield!crystals!of!wild
<
type!
Rma#
cyt!
c
#
(
7
)
.
!
Crystals!were!grown!against!a!1
<
mL!reservoir!with!mother!liquor!comprised!of!2.
0
!
μL!of!14.0!mg/mL!
Rma#
TDE!
(in!20!mM!Tris!HCl,!
pH!7.5)!
and!1.0!μL!of!well!solution.!
Very!small!crystals!were!observed!after!two!weeks!in!1.8
<
1.9!
M!(NH
4
)
2
SO
4
,
!
2%!isopropanol.!Further!
optimization
!
failed!to!yield!larger!crystals,!but!showed!that!
the! protein!
crystallized
!
within
!
the! range! of
!
1.4
<
1.9! M! (NH
4
)
2
SO
4
,! 1
<
3%! isopropanol.! Additive!
screening!was!performed!
using!an!additive!screening!kit!(Hampton!Research
,!Aliso!Viejo,!CA
)!
and!with!mother!liquor!comprised!of!2.0!μL!of!14.0!mg/mL!
Rma#
TDE,!1.0!μL!of!well!solution!(1.7!
M!(NH
4
)
2
SO
4
,!1.5%!isopropanol),!and!0.2!μL!of!additive!solution.!Additive!screening!showed!that!
addition!of!NaSCN!yielded!larger!and!more!
numerous!
crystals.!Further!fine!screening!around!this!
condition!revealed!that!the!best!crystals!were!obtained!using!1.43
<
1.48
!
M!(NH
4
)
2
SO
4
,!300
<
400!
mM!NaSCN,!and!1.5%!isopropanol!(2.0!μL!of!14.0!mg/mL!
Rma#
TDE,!1.0!μL!well!solution).
!!
Under!
these!co
nditions,!crystals!formed!over!3
<
5!days,!and!reached!maximum!size!after!7
<
10!days.!
Crystals! were! cryo
<
protected! by! addition! of! ethylen
e! glycol! to! the! well! solution! to! a! final!
concentration!of!20%.!Crystals!were!flash!frozen!and!stored!in!liquid!N
2
!
until!diffraction.
!
For!crystals!soaked!with!the!carbene!precursor!Me
<
EDA,!the!mother!liquor!was!supplemented!
with!1
!
M!Me
<
EDA!in!isopropanol!to!a!final!concentration!of!40!mM!Me
<
EDA.!!Crystals!were!soaked!
at!room!temperature!for!30!minutes!before!moving!the!samples!to!4ºC.!!At!4ºC,!the!crystals!were!
soaked!for!approximately!1!minute!in
!
a!solution!of!mother!liquor!supplem
ented!with!40!mM!Me
<
EDA!and!50!mM!sodium!dithionite!before!
flash
<
freezing!in!liquid!nitrogen
.!!
Prolonged!soaking!in!
either!of!the!Me
<
EDA!supplemented!solutions!caused!the!crystals!to!dissolve.!!
!
Diffraction!data!were!collected!remotely!at!the!Stanford!Sync
hrotron!Radiation!Laboratories!on!
beamline!12
<
2
,!with!a!wavelength!of!0.97946!Å,!and!at!a!temperature!of!100!K
.!Crystals!routinely!
diffracted!at!or!below!
1.7
!
Å,!and!the!data!were!integrated!and!scaled!using!XDS
!
(
8
)
!
and!AIMLESS
!
(
9
)
.
!
A!resolution!cutoff!of!
I/
"
!
>!2.5
!
was!applied!along!
all
!
ax
es
!
of!diffraction.
!
These!data!contributed!
to!model!quality!as!judged!by!R
free
!
in!the!final!bin!<!0.
27
.!The!structure!was!solved!using!molecular!
replacement!with!PHASER
!
(
10
)
,!as!implemented!in!CCP4
!
(
11
)
.!For!
Rma#
TDE,!t
he!search!model!
comprised!a!single!monomer!of!
wild
<
type!
Rma#
cyt!
c
!
(PDB!ID:!
3CP5
)!(
12
)!subjected!to!10!cycles!
of!geometric!idealization!in!Refmac5
!
(
13
)
!
and!removal!of!all!ligands.!
For!carbene
<
bound
!
Rma#
TDE,!the!search!model!comprised!a!single!monomer!of!
Rma#
TDE!(PDB!ID:!
6CUK)!
subjected!to!
10!cycles!of!geometric!idealization!in!Refmac5!and!removal!of!all!ligands.!
Model!building!was!
performed!in!Coot
!
(
14
)
!
beginning!with!data!processed!at!2.2!Å,!followed!by!subsequent!inclusion!
of!increasingly!higher!resolution!s
hells!of!data!with!relaxed!geometric!constraints.!Refinement!
was!performed!using!
Refmac5
.
!
The!MolProbity!server
!
(
15
)
!
was!used!to!identify!rotamer!flips!and!
clashes
.!
After!the!protein,!ligand,!and!solvent!atoms!were!built,!TLS!operators!were!added!to!
refinem
ent
!
(
16
)
,! which! resulted! in! substantial! improvements! in! R
free
!
for! the! models.!
Crystallographic! and! refinement! statistics! are! reported! in!
Table! S1
.!
The! carbene! ligand! was!
modelled!in!
a!single!conformation!
at!100%!occupancy,!which!resulted!in!the!atoms!
comprising!
the!Fe
–
C!bond!to!have!B
<
factors!of!14.74!and!21.36
,
!
respectively.!Occupancy!was!not!refined!by!
B
<
factor!matching,!as!the!
electron!density!suggested!only!a!single!conformation,!and!
B
<
factors!
on!both!atoms!were!both!sufficiently!low!and!similar!to
!
indicate!high!occupancy!in!the!crystal.
!
The
!
carbene
<
bound!
Rma#
TDE
!
structure!is!deposited!with!PDB!ID:!
6CUN,!
and!the!
Rma#
TDE!structure
!
without!the!carbene!ligand
!
is!deposited!with!PDB!ID:!
6CUK.!!
Analysis!of
!
the!protein!structures!
with!MolProbity!showed!tha
t!the!carbene
<
bound!
Rma#
TDE!structure!had!a!Mo
lProbity!score!of!
1.0,!and!the!
Rma
!
TDE!structure!had!a!MolProbity!score!of!0.9,!putting!both!structures!in!the!
highest!percentile!rank!for!MolProbity!scores.!The!carbene
<
bound!
Rma#
TDE!structure!contains!
!
8
!
98.15%!
Ramachandran
<
favored
!
rotamers,!with!no!Ramachandran!outliers,!and!the!
Rma
!
TDE!
structure!contains!99.12%!Ramachandran
<
favored
!
rotamers!and!no!Ramachandran!outliers.
!
!
!
!
(
I
)(Mössbauer(spectroscopy
(
(
General(Description(of(Mössbauer(Spectroscopy:
(
Mössbauer!spe
ctra!were!recorded!on!a!spectrometer!from!SEE!Co.!(Edina,!MN)!operating!in!the!
constant!acceleration!mode!in!a!transmission!geometry.!The!sample!was!kept!in!an!SVT
<
400!
cryostat!form!Janis!(Wilmington,!MA).!The!quoted!isomer!shifts!are!relative!to!the!centr
oid!of!the!
spectrum!of!a!metallic!foil!of!α
<
Fe!at!room!temperature!(RT).!Solution!samples!were!transferred!
to!a!sample!cup!and!chilled!to!77!K!inside!of!the!glovebox,!and!unless!noted!otherwise,!quickly!
removed!from!the!glovebox!and!immersed!in!liquid!N
2
!
until!mounted!in!the!cryostat.!Data!analysis!
was! performed! using! version! 4! of! the! program! WMOSS! (www.wmoss.org)! and! quadrupole!
doublets!were!fit!to!Lorentzian!lineshapes!except!where!noted.!See!discussion!below!for!detailed!
notes!on!the!fitting!procedure.
!
!
Sample(preparation(for(Mössbauer(Spectroscopy:
(
Sample! preparation! conditions! for! Mössbauer! samples! were! designed! to! be! similar! to! those!
conditions!used!to!assay!the!biocatalytic!activity!of!
Rma#
TDE,!as!shown!in!Supplementary!Figure!
4F.!!
!
!
In!an!anaerobic!chamber,!an!aliquot!of!
57
Fe
<
label
l
ed!purified!
Rma#
TDE!(4
.
00!
m
M)!was!thawed.!
For!the!reduced!
Rma#
TDE!sample,!341!μL!of!protein
!
(
4.00!mM!in!20!mM!NaPi,!pH!7.5
)
!
was!added!
to!a!Mössbauer!cup!containing!3.7!mg!sodium!dithionite.!
Acetonitrile!(
18
!
μL
)
!
was!added!to!the!
Mössbauer! cup,! and! the! sample! was! pipetted! up! and! down! to! ensure! the! sample! was!
homogenous!(approximately!10!seconds)
.!!
The!sample!was!then!flash
<
frozen!on!liquid!nitrogen.
!
Final! concentrations:! 3.8! mM! protein,!
19
!
mM! NaPi! (pH! 7.5),! 50
!
mM! sodium! dithionite,! 5%!
acetonitrile.
!
!
For!the!reduced!Me
<
EDA
<
treated!
Rma#
TDE!sample,!341!μL!of!protein!
(
4.0
!
mM!in!20!mM!NaPi,!
pH!7.5
)!
was!added!to!a!Mössbauer!cup!containing!3.7!mg!sodium!dithionite.!18!μL!of!1.0!M!Me
<
EDA!in!acetonitrile!was!added!to!th
e!Mössbauer!cup,!and!the!sample!was!pipetted!up!and!down!
to!ensure!the!sample!was!homogenous!(approximately!10!seconds).!The!sample!was!then!flash
<
frozen!on!liquid!nitrogen.
!
Final!concentrations:!3.8!mM!protein,!
19
mM!NaPi!(pH!7.5),!50!mM!
sodium!dithionite,
!
50!mM!Me
<
EDA,!5%!acetonitrile.
!
!
!
(
J
)(Electron(paramagnetic(resonance(
(EPR)(
spectroscopy
(
(
General(Description(of(EPR(Spectroscopy:
(
EPR!spectra!were!recorded!on!a!
Bruker!EMX!X
<
band!CW
<
EPR!Spectrometer
!
(Bruker,!Billerica,!
MA)!at!4!K.
!
using!Bruker!Win
<
EPR!software!(ver.!3.0).!
Data!were!collected!with!
in!parallel!mode!
with!a!power!of!10!mW,!and!in!perpendicular!mode!with!a!power!of!2!mW.
!
!
(
Sample(preparation(for(
EPR(spectroscopy
:
(
In!an!anaerobic!chamber,!an!aliquot!
of!
purified!
Rma#
TDE
!
wa
s!thawed
!
and!diluted!to!
1.00!mM
!
with!20!mM!NaPi,!pH!7.5!with!10%!v/v!DMSO
.!
Solid!sodium!dithionite!was!added!to!the!purified!