Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging.

Abstract

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ⇆ H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.

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Published: September 14, 2011
r2011 American Chemical Society
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pubs.acs.org/JACS
Catalytic Turnover of [FeFe]-Hydrogenase Based on Single-Molecule
Imaging
Christopher Madden,†Michael D. Vaughn,†Ismael Díez-P? erez,‡,§Katherine A. Brown,^Paul W. King,^
Devens Gust,*,†Ana L. Moore,*,†and Thomas A. Moore*,†
†Center for Bioenergy and Photosynthesis, Center for Bio-Inspired Solar Fuel Production and Department of Chemistry and
Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, United States
‡Center for Bioelectronics and Biosensors, Biodesign Institute and Department of Electrical Engineering, Arizona State University,
Tempe, Arizona 85287, United States
§Department of Physical Chemistry, University of Barcelona, Barcelona, Spain 08028
^Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States
b
S Supporting Information
’INTRODUCTION
Hydrogenasesareofgrowinginterestduetotheirutilizationof
common metals at their active sites and ability to catalyze the
2H++ 2e?/ H2redox system under nearly activationless
conditions, withconcomitantenergystorage or release.1?5They
serve as models for catalysis of this most fundamental of redox
reactions. Interest in the catalytic abilities of hydrogenases for
hydrogen generation has led to various electrochemical studies6?9
as well as photoelectrochemical hydrogen production studies.10
A typical [FeFe]-hydrogenase is CaHydA, a 65.4 kD protein
with a highly conserved [6Fe-6S] catalytic H-cluster as well as
three [4Fe-4S] and one [2Fe-2S] accessory clusters. The proxi-
mity of the distal [FeS] cluster to the exterior of the protein
allows electrons to be transferred directly from an external redox
partner, presumably through a chain of [FeS] clusters, to the
H-cluster, where catalysis occurs and hydrogen is produced or
oxidized.
Direct electrochemical measurement of catalytic currents on
macroscopic electrodes as a function of applied potential has
yielded valuable information.4The CaHydA [FeFe]-hydroge-
nase, as well as other [FeFe]-hydrogenases, reportedly show a
preference for proton reduction over hydrogen oxidation,5
leading them to be investigated as a means of producing
hydrogen. On the other hand, hydrogenases biased toward
hydrogen oxidation could serve as catalysts in fuel cells.11The
mechanism of bias in either hydrogen oxidation or proton
reduction is not clear: electrochemical studies have shown that
these catalysts appear to operate very near the prevailing
thermodynamic potential of the H+/H2couple and therefore
cannot differentially favor the reduction or oxidation.
Numerous electrochemical studies with both [NiFe]- and
[FeFe]-hydrogenases have been performed at carbon elec-
trodes,8,10,12?16while a handful have been performed on gold
and modified gold electrodes.17,18Although most studies utilize
hydrogenaseadsorbedonanelectrode asanensembleaverage of
orientations,19attemptshavebeenmadetospecificallyorientthe
protein on the electrode surface.20,21A suitably oriented mole-
cule allows for better defined interfacial electron transfer22and
thepossibilityofobservingthelargestcurrentdensitiesattainable
Received:August 15, 2011
ABSTRACT: Hydrogenases catalyze the interconversion of protons and hydrogen
according to the reversible reaction: 2H++ 2e?/ H2while using only the earth-
abundant metals nickel and/or iron for catalysis. Due to their high activity for proton
reduction and the technological significance of the H+/H2half reaction, itis important to
characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and
electrochemical techniques.Followingadetailedelectrochemicalandphotoelectrochem-
ical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now
report electrochemical and single-molecule imaging studies carried out on a catalytically
active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of
the [FeFe]-hydrogenase from Clostridium pasteurianum, CpI, was adsorbed to a nega-
tively charged, self-assembled monolayer (SAM) for investigation by electrochemical
scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging
revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other
electrochemical investigations. Cyclic voltammetryyieldedacharacteristic cathodic hydrogen production signal whenthe potential
was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with
macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency
(TOF) as a function of potential for single [FeFe]-hydrogenase molecules.

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for a given system. Maximum current densities are necessary to
observe maximum turnover, and the number of molecules
participating in the reaction must be known in order to calculate
the TOF. Previously, surface coverage of hydrogenases on
electrodes was either estimated on the basis of the electroactive
surfaceareaofanelectrode, orbyinhibitingtheenzymetoreveal
nonturnover signals from redox cofactors inside the protein.23,24
Whilethelattertechniquecanbeusefulandhasprovidedthefirst
measurements of electroactive surface coverage, it has only been
demonstrated in a limited number of cases with specific hydro-
genases, most notably a [NiFe]-hydrogenase from Allochroma-
tium vinosum.25Calculation of the electroactive surface coverage
of enzyme enabled Armstrong et al., to calculate accurate TOFs
for various hydrogenases.7,11One study has also shown that a
[NiFe]-hydrogenase from Thiocapsa roseopersicina can be im-
aged with a scanning tunneling microscope in a Langmuir?
Blodgett film; I?V curves of the protein film were reported.26
The ability to measure redox processes of simple proteins
through SAMs has also been investigated previously where
one-electron oxidations and reductions were taking place.27
More complicated, multielectron or catalytic processes have also
been measured through SAMs. However those studies did not
involve the use of single-molecule topographic techniques such
as STM to determine surface concentrations.17,28Knowing the
actual surface coverage on an atomically flat surface is crucial to
accurately calculate the catalytic TOF, measured as a function of
applied potential, per single adsorbed enzyme molecule.
The measurement of the maximum TOF of hydrogenase
molecules is the mostdirectindicationof catalyticprowess.Such
ameasurementrequiresaccurateknowledgeoftheconcentration
of active protein and of substrates and products. However,
performing this measurement in solution remains a formidable
challenge because saturating concentrations for both substrates
cannot be realized. Varying the substrate (H+) concentration as
necessary to allow for a typical biochemical assay to determine
the turnover of the hydrogenase is not possible because the
protein is rendered inactive outside a narrow pH range.10,29,30
In this study, we investigate the enzymatic turnover of an
[FeFe]-hydrogenase, CaHydA from Clostridium acetobutylicum,
which is homologous (70% identity)31with the [FeFe]-hydro-
genase from C. pasteurianum, CpI, for which structural data
exists.32,33The CaHydAwas chosen forthis study due toits high
catalytic activity for hydrogen production and the relative ease
of recombinant expression.31The enzyme was adsorbed onto a
negatively charged SAM on a flat gold surface for investigation
by electrochemical scanning tunneling microscopy (EC-STM)
techniques and macroscopic electrochemical measurements.
The EC-STM imaging allowed quantitative determination of
the number of bound enzyme molecules. The bound enzymes
were catalytically active, and cyclic voltammetry (CV) yielded a
characteristic cathodic hydrogen production signal when the
potential was scanned sufficiently negative. The quantitative
determinationoftheenzymedistributionontheAu-SAMsurface
coupled with macroscopic electrochemical measurements ob-
tainedfromthesameelectrodeallowedtheevaluationofaTOFasa
function of potential for single [FeFe]-hydrogenase molecules.
’EXPERIMENTAL SECTION
Electrochemical scanning tunneling microscopy (EC-STM) studies
were performed with a Pico-SPM (Agilent, AZ) using a Nanoscope E
controllerandabipotentiostat(Agilent,AZ).Imagingexperimentswere
performed in a homemade Teflon cell using as a working electrode an
atomically flat Au (111) substrate prepared by thermally evaporating
∼130 nm of gold (Alfa Aesar 99.999%) onto freshly cleaved mica
surfacesunderultrahighvacuum(∼2?10?8Torr).TheSTMtipswere
prepared by mechanically shearing Pt/Ir wire (80/20, 0.25 mm dia-
meter)andcoatingthetipswithApiezonwax.Alltipshad<1pAleakage
current,thetunnelingcurrentsetpointwas200?400pAwitha10nA/V
currentamplifier,anda100mVsamplebiaswastypicallyapplied.AllAu
(111) substrates were annealed with a hydrogen flame prior to use and
subsequently immersed in ethanolic solutions of mercapto-carboxylic
acids to form self-assembled monolayers (SAMs). The resulting SAMs
were imaged without protein to ensure formation of the SAM and
cleanliness of the surface. The CaHydA was subsequently adsorbed
in situ to the monolayer with 0.1 M phosphate buffer, pH 7.0, as the
supporting electrolyte and imaged in EC-STM mode to control
substrate potential. All EC-STM images were recorded with a substrate
potential of ?400 mV vs Ag/AgCl. Titration of protein to control
surfacecoverageresultsintheadsorptionofproteintothemodifiedgold
surface over the course of several minutes to a few hours to reach
equilibrium.
The [FeFe]-hydrogenase CaHydA from C. acetobutylicum was pur-
ified and expressed in Escherichia coli and assayed for H2evolution
activity according to previously reported procedures.34Due to the
extreme oxygen sensitivity of [FeFe]- hydrogenases,9,35,36all work was
performed in an anaerobic chamber (Coy Laboratory Products) under
strictly anaerobic conditions (2?3% H2, bulk N2, <1 ppm O2). Under
anaerobicconditionsandatambienttemperature(∼25?C),CaHydAis
stable for several hours in the electrochemical STM setup with little or
no observed loss in electrocatalytic activity. The specific activity units
(U) are defined as 1 μmol H2produced min?1mg?1of enzyme from
sodium dithionite reduced methyl viologen (7.5 mM) as an electron
donor.ThisstudyusedtwoseparateCaHydApreparationswithnominal
solution-basedspecificactivitiesforH2productionof177and280Umg?1,
which correspond to TOFs of192 s?1and 303 s?1respectively. All data
were normalized to both the surface coverage ofprotein and the specific
activity.
Cyclic voltammetry was performed with a CH Instruments 650C
electrochemical workstation using a platinum wire counter electrode
and either a silver wire quasi reference or a silver/silver chloride refer-
ence electrode. The Au (111) substrate modified with a carboxylate-
terminatedSAMwasusedasaworkingelectrode.AllCVwasperformed
in the same EC-STM cell, with a geometric surface area of 0.283 cm2.
The same sample was used for imaging by EC-STM inside an anaerobic
chamber as well as anaerobic CV in a typical three-electrode configura-
tion. All CVs were recorded in 0.1 M phosphate at pH 7.0 with a scan
rate of 50 mV/s.
STM images were scrutinized by eye to estimate the number of
proteinsonthesurface.Several100nmby100nmimagesfromdifferent
areas of the electrode were analyzed, particles were counted, and an
average coverage was obtained, which was then used to estimate the
number of proteins on the geometric surface. Variation in surface
coverage among images was <10% for a given sample. To verify the
methodology of counting individual particles in order to quantify
enzymes on the electrode, two of the STM image-quantified samples
were tested for iron content via a ferrozine iron binding assay adapted
from previously reported procedures.37,38The quantity of iron deter-
mined by this procedure differed from that determined by counting the
images by 20?50% underestimation, indicating that qualitatively the
method of counting the images is valid. Due to the minute amounts of
protein in the experiments, (∼10?12mol of protein/cm2), the difficulty
of recovering protein from surfaces, and detection limitations by
UV?vis absorption measurements, data from the STM images alone
wereusedtocalculateaTOFinthisreport.Recoveredsampleswerealso
analyzed by SDS-PAGE to confirm that the molecular weight of the

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adsorbed protein was consistent with CaHydA (see Supporting
Information).
’RESULTS
Assembly on SAMs. SAMs were prepared on atomically flat
goldelectrodeswithcarboxylic-acidterminatedalkanethiols.The
acid moieties, which are negatively charged at the pH of these
experiments, are designed to interact with the large regions of
positive charge that are present on the protein surface. Ideally,
such interactions are both strong and serve to orient the enzyme
molecules all with the same face toward the gold. We employed
SAMs having 3?11 carbon atoms in order to alter the distance
between the protein and the electrode surface. Figure 1a?c
depicts the proposed interaction between the positively charged
protein surface and the negatively charged monolayer surface
based on electrostatic considerations.39Although, the relative
energies of this orientation and others were not rigorously
calculated; the area of exposed positive charge of CaHydA inter-
acting with the carboxylated surface of the SAM is maximized in
these structures. Recently, Brown et al. have shown that CdTe
nanocrystalscappedwith3-mercaptopropionicacidinteractwith
a region of positive charge on the surface of CaHydA to form a
stable complex for the photoproduction of hydrogen.40Our
system presumably utilizes the same region of positive charge,
which surrounds the proposed binding site for the negatively
charged ferredoxin during in vivo electron transfer.31This is the
point where electrons are thought to enter the CaHydA electron
transfer chain. Cyclic voltammetric studies were performed on
these Au-SAM electrodes with bound CaHydA and large cata-
lytic electrochemical signals for hydrogen production were
observed (Figure 1d), suggesting that the orientation of the
protein on the electrode is favorable for electron transfer (ET).
EC-STM. EC-STM was chosen for imaging in this work for its
ability to provide high-resolution imaging in a liquid medium as
well as potentiostatic control of both tip and substrate. With
CaHydA stably adsorbed onto carboxylate-terminated SAMs,
the resulting surface was imaged, revealing random, relatively
uniform coverage (Figure 2a?d). The electrochemical signal
(Figure 1d) on the Au?SAM electrodes resembles that on PGE
electrodes,10both in observed current density for short carbon
chains, and waveform. It has been previously demonstrated that,
under conditions like these, hydrogenase molecules free in
solution do not contribute significantly to the catalytic
current.30,41This suggests that the enzyme?surface interaction
is stable and that the catalytic current can be ascribed to the
enzyme molecules detected in the STM images.
The apparent height of the protein on the surface reflects the
magnitude of the current flowing through the enzyme between
the tip and gold substrate. This current changes with substrate
potential (EC-STM) due to the properties of the redox active
cofactorsincorporatedintheprotein,specificallythedistal[FeS]
cluster which serves as an initial acceptor for intramolecular ET.
As the substrate potential is shifted negative, the Fermi levels of
both working electrodes (i.e., the tip and substrate) will bracket
the redox level of the protein, at some point meeting resonant
Figure1. (a)HomologymodelofCaHydA[FeFe]-hydrogenase(seeSupportingInformation)ona6-mercaptohexanoicacid-modifiedgoldelectrode.
Panels a?c are shown for identical orientations. (b) Electrostatic surface potential of homology model of CaHydA on negatively charged SAM surface.
(c)IsopotentialsurfacesgeneratedbyCaHydA.The+1kcal/(mol3e)surfaceisdepictedinblue,andthe?1kcal/(mol3e)surfaceisdepictedinred.The
orientationdepictedinthispanelmaximizesthepositivesurfaceexposuretothecarboxylatesoftheSAM,althoughotherorientationsarepossible.(b,c)
electrostatic calculations were performed at neutral pH and room temperature (25 ?C). (d) Typical cyclic voltammogram of CaHydA on a
6-mercaptohexanoic acid-modified gold electrode. The black scan is a blank of the 6-mercaptohexanoic acid-modified electrode. The red scan depicts
CaHydA adsorbed to the SAM.

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tunneling conditions and giving rise to an increase in the tun-
neling current through the protein. A higher current is observed
at roughly ?0.6 V vs Ag/AgCl, the potential at which proton
reduction occurs, which is closely matched to the redox proper-
ties of the [FeS] clusters. Under these conditions, topographical
STM images show larger apparent heights for the redox active
hydrogenase (see Figure 2b?d). When the Fermi levels of the
electrodes are positive or negative of the midpoint of the redox
center (i.e., nonresonant tunneling conditions), the height
appears smaller due to the decreased protein conductivity.
Theoretically this observation should give a Gaussian distribu-
tion ofheights versussubstrate potential as thepotentialisswept
past the midpoint of the redox center. This phenomenon has
been recorded previously for redox proteins such as azurin42as
wellasotherredox-activesmallmolecules.43Inourcase,onlyone
side of the curve can be observed due to limits imposed by
solvent reduction at the tip at increasingly negative potentials.
Cyclic Voltammetry. The overall electrochemical properties
of such electrodes may be studied using standard cyclic voltam-
metric techniques. A catalytic hydrogen production current due
toenzymaticturnoverisobservedwhensufficientdrivingforceis
applied to the electrode as seen in Figure 3a. For accurately
measuring these currents, samples with high-density surface
coverage (0.5?2 pmol/cm2) were prepared as seen in the inset
of Figure 3a. The observed catalytic current is directly related to
the amount of hydrogen produced from the enzyme. Taken
together, the catalytic current and surface coverage (Figure 3a
inset) were used for the calculation of a TOF per adsorbed
CaHydA. The distance between the electrode and enzyme was
altered by varying the SAM alkyl chain length from three to
elevencarbons.Asaresult,thecurrentdensitiesatagivenvoltage
declined exponentially with increasing SAM length. Figure 3b
depicts typical CVs of CaHydA on Au?SAM electrodes of
lengths from three to eleven carbons, showing a decreasing
catalytic current (i.e., turnover) for increasing chain length.
’DISCUSSION
From the catalytic hydrogen production current recorded
through SAMs of various lengths and the number of enzyme
molecules per electrode area counted from the STM images of
eachelectrode,aTOFpermoleculeofCaHydAcanbecalculated
at any given applied potential. The TOF per protein at ?0.7 V
was plotted as a function of SAM length and fitted to an
exponential decay function (Figure 3c) according to eq 1, where
I is the tunnel current at a distance d from the electrode, I0is the
limiting current in the absence of the SAM layer, and β is the
electronic decay constant.
I ¼ I0e?βd
Anincrementaldistancepercarboninthealkylchainof1.25Å
wascalculatedonthebasisof109.5?carbon?carbonbondangles
and a carbon?carbon bond length of 1.54 Å. From this, we
calculated the length of the SAM alkyl chain and estimated the
distance (d) through which electron transfer between the
enzyme surface and the electrode must occur. The experimental
electronicdecayconstantwasdeterminedtobe0.82(0.16Å?1.
This agrees well with values reported in the literature for self-
assembled monolayers of alkanethiols and is consistent with
values obtained from drastically different techniques, including
ð1Þ
Figure2. (a)EC-STMimageofa3-mercaptopropionicacid/ethanethiolSAM(1:1)onaAu(111)surfacewithoutprotein;Ebias=200mV,It=400pA.
(b?d) CaHydA adsorbed onto the modified surface at substrate potentials ranging from ?400 mV to ?600 mV vs Ag/AgCl. The images show an
increasing apparent height with increasingly negative potentials. (e) Graph showing the apparent height of CaHydA as a function of potential.

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single-molecule junction measurements.44The exponential de-
cay behavior is a consequence of control of the catalytic rate by
ETthroughtheSAMtotheprotein.ETthroughtheproteinitself
to the catalytic site is the same for all SAM-protein constructs
and, we conclude, does not control the catalytic rate at the
potentials investigated.
Extrapolationofthisplottoadistanceofzero,approximatinga
CaHydA molecule in direct contact with the bare gold electrode
surface where eq 1 simplifies to I = I0, gave a TOF of ∼21,000 (
12,000 s?1at pH 7.0, which is higher than previous estimates in
the literature for hydrogenases.11,28,29Methods using nonturn-
over signals for estimates of protein coverage have given a TOF
upward of 10,000 s?1.4Catalytic current normalized by STM-
derived surface density thus provides an average of the TOF for
individual enzyme molecules for any given potential within the
window of the electrochemical signal for CaHydA. This number
doesnotaccountforinactive proteinattheelectrodeinterface or
enzymes in an orientation unfavorable for interfacial ET and is
thus a lower limit for turnover.
The electrochemical signal observed in cyclic voltammetry
and the potential at which proton reduction occurs agree well
with those reported in the literature for various electrode
surfaces,10,17,20confirming that the kinetics of ET through the
protein are not altered in this system. Theoretically, a limiting
cathodic current should be observed when the rate of enzymatic
proton reduction is slow compared to the rate of interfacial ET
from the electrode to the protein. When these conditions are
satisfied, the limiting current is directly related to the maximum
turnover of the enzyme for the reduction of protons at a given
pH. As the CVs indicate, even at the relatively low [H+] used in
our experiments, a limiting current was not observed. This may
show that at these currents, a maximum turnover was not
reached. There are other explanations for the observed lack of
limiting currents. In one interpretation, a dispersion of nonspe-
cific electrostatic protein?surface interactions (see Figure 1),
such as could be the case with our system, preclude the direct
observation of a limiting current.22
As a complement to the STM images, additional measures
were taken to ensure the identity and quantity of CaHydA. To
confirm the identity of the protein adsorbed to the surface, SDS-
PAGE was run with samples recovered from the Au (111)
substrates used for STM imaging experiments against purified
CaHydA. Matching bandswere observedat 65 kD (see Support-
ing Information). The detection of this small amount of protein
was also consistent with the STM images in that three samples
were combined for the SDS-PAGE, which was just sufficiently
abovethelimitofdetectionforvisualizationbysilverstaining.45?47
As an independent estimate of amount the CaHydA present on
the STM substrate, samples were recovered under denaturing
acidic conditions, and the Fe was quantified using a modified
ferrozine assay.37,38Although this assay was near its limit of
detection, the amount of Fe observed was also qualitatively
consistent with the STM imaging results (see Supporting
Information).
’CONCLUSION
We have designed a method for immobilizing an [FeFe]-
hydrogenase, CaHydA, on an atomically flat gold electrode for
topographic and electrochemical experiments on catalytically
active samples. Direct observation of immobilized protein via
EC-STM techniques shows the actual surface coverage which,
when coupled with macroscopic electrochemical analysis, allows
calculation of TOF as a function of potential at the single
molecule level. From our analysis, we observe a TOF of
∼1000 s?1for CaHydA on a three-carbon SAM, and when
extrapolated to bare gold, the TOF is estimated to be ∼21,000
s?1at ?0.7 V vs Ag/AgCl. Using a bipotentiostat, resonant
tunneling at potentials consistent with the redox midpoints
of cofactors within the protein was observed. The β-value of
0.82Å?1forelectrontransferthroughalkanethiolSAMsmeasuredvia
determinationofcatalyticTOFisconsistentwithvaluesobtained
from radically different techniques, such as STM break junction.
Taken together, the number of molecules calculated from the
images,theexponentialfittothenumberofcarbonsintheSAMs,
the value of β, and the indication of the redox activity for FeS
centers form a consistent picture of electrocatalytic activity of
CaHydA on SAMs. However, these results do not fully address
thequestion ofheterogeneityintheenzyme’scatalyticactivityor
interaction with the electrode. The stability of immobilized
CaHydA on SAMs under these conditions augurs well for future
experiments to explore catalytic detail at the single molecule level.
’ASSOCIATED CONTENT
b
S
Supporting Information.
and CVs and STM images. This material is available free of
charge via the Internet at http://pubs.acs.org.
Extensive experimental details
’AUTHOR INFORMATION
Corresponding Author
tmoore@asu.edu
Figure 3. (a) Cathodic voltammogram of a high surface coverage CaHydA electrode. (Inset) STM image of the same electrode. Combining the
voltammogramandproteincoveragefromtheinset allowsforcalculation ofaTOFper adsorbedproteinmolecule. (b)EnzymaticturnoverofCaHydA
recordedondifferentlengthsofcarboxylate-terminatedSAMsasafunctionofappliedpotential.Ratedataaredeterminedforthecathodicscanat?0.7V
vs Ag/AgCl, as indicated as a dashed line. (c) Single-protein turnover as a function of number of carbons in the SAM fit to an exponential function.

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’ACKNOWLEDGMENT
We thank Professor Michael Hambourger for insightful dis-
cussions and helpful advice and Professor N. J. Tao for use of
laboratory equipment. C.M. thanks Science Foundation Arizona
forfinancialsupport.I.D.P.thankstheMarie-CurieInternational
Outgoing Fellowship within the seventh European Community
Framework and the MICINN Ramon y Cajal programs for
financial support. M.V. is supported by the Graduate Research
Fellowship Program from the National Science Foundation.
K.A.B. and P.W.K. gratefully acknowledge funding by the U.S.
DepartmentofEnergy,DivisionofChemicalSciences,Geosciences,
and Biosciences, Office of Basic Energy Sciences; and support
by the U.S. Department of Energy under Contract No.
DE-AC36-08-GO28308 with the National Renewable Energy
Laboratory.
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