ElectronicSupplementaryInformationfor:

Nitrogenasebioelectrocatalysis:heterogeneousammoniaandhydrogenproductionbyMoFeprotein

by

RossD.Milton,SofieneAbdellaoui,NimeshKhadka,DennisR.Dean,DónalLeech,LanceC.SeefeldtandShelleyD.Minteer

Electronic Supplementary Material (ESI) for Energy & Environmental Science.This journal is © The Royal Society of Chemistry 2016

SupplementaryFiguresandTables

Figure S1 – Cyclic voltammetry of a BSA control bioelectrode in the absence (dashedline) andpresence (solid line) of 50mMNO2

-, evaluatedat a scan rateof 2mv s-1 inHEPESbuffer(pH7.4,250mM)intheabsence(redlines)orpresence(blacklines)of200µMCc+.

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FigureS2-Cyclicvoltammetryofadditionalcontrolbioelectrodesinthe(a)absenceorpresence of 50mM (b)N3

- or (c)NO2-, evaluated at a scan rate of 2mv s-1 inHEPES

buffer (pH 7.4, 250 mM). Complete MoFe protein bioelectrodes (prepared with WTMoFeprotein)arecomparedagainstbioelectrodeswithoutCc+/Cc (blackdashed line),without polymer and MoFe protein (black dotted line), as blank glassy carbon (GC)electrodesonly(redsolidline)orwithoutCc+/CcandMoFeprotein(reddashedline).

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cComplete WT MoFe bioelectrode

Polymer + WT MoFe protein (no Cc+)

GC electrode + Cc+ (no polymer/MoFe protein)

GC Electrode only (no polymer/MoFe protein/Cc+)

Polymer (no Cc+/MoFe protein)

Figure S3 – Bulk bioelectrosynthetic reduction of 50 mM (a) N3

- and (b) NO2- by β-

98TyràHisMoFeproteinbioelectrodes(blacklines),operatinginstirredHEPESbuffer(pH7.4, 250mM) containing200µMCc+ (Eapplied = -1.25Vvs. SCE). Control bioelectrodes(redlines)werepreparedusingapo-MoFeprotein(redlines).Substrateswereinjectedat120s.

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FigureS4–CalibrationstandardsforNH3usingortho-phthalaldehyde(detailedbelow,n

=3).

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Table S1 – Catalytic current densities extracted from cyclic voltammograms ofrespectiveMoFeproteinbioelectrodes(-1.35Vvs.SCE)operatingat2mVs-1inHEPESbuffer (pH 7.4, 0.2 M) containing 200 µM Cc+ (n = 3, mean ± SD). Catalytic currentdensitiesarereportedcorrectedtocorrespondingcurrentdensitiesobtainedfromapo-MoFeproteincontrolbioelectrodes(errorspropagated).

MaterialsandMethodsChemicalsAll chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless specifiedotherwise. Poly(vinylamine) and ethylene glycol diglycidyl ether (EGDGE) werepurchasedfromPolySciences,Inc.Saturatedcalomel(SCE)referenceandglassycarbonworkingelectrodeswerepurchasedfromCHInstruments,Inc.ElectrochemicalanalysisAll electrochemical analyses were performed anaerobically (<1 ppm O2) to avoidcompetitiveO2 reductionat theworkingelectrodeand topreservenitrogenaseMoFeprotein activity. HEPES buffer (pH 7.4) was used as the supporting electrolyte at aconcentration of 250 mM to avoid migration issues associated with N3

- and NO2-.

Electrochemical measurements were performed using a CH Instruments (Inc.)potentiostat (1230b),utilizinga largePt counterelectrode ina traditional3-electrodeconfiguration.CellgrowthandnitrogenaseMoFeproteinpurificationAzotobacter vinelandii strainsDJ995 (wild-typeMoFe protein) andDJ1003 (apo-MoFeprotein) were grown and polyhistidine-tagged proteins were purified as previouslyreported.1Similarly,strainDJ939(β-98Tyr→His)wasgrownandthecorrespondingnon-Histagged MoFe protein was purified as described. Protein manipulation was eitherperformedunderanargonatmosphereinseptum-sealedvials(usingaSchlenkline)orwithinananaerobictent(<1ppmO2,CoyLaboratoryProducts,MI,USA).

NitrogenaseMoFeproteinswereassayedforH+,C2H2andN2activity. nmol/min/mgMoFeprotein

MoFeProtein H2 C2H4 NH3DJ995 2493 2349 606DJ939 1100 600 220

MoFeproteinbioelectrodepreparationPurifiedMoFeprotein(20mgmL-1,15µL)wasthawedanaerobicallyandimmobilizedatthesurfaceofaglassycarbonelectrodebymixingwithpoly(vinylamine)(10mgmL-1,15µL)andEGDGE(10%v/v,2µL);5µLofthismixturewasappliedtothesurfaceofeachglassycarbonelectrodeanddriedunderreducedhumidityfor1hour,priortotesting. Theratioofpolymer:crosslinkerisofimportance;toolittlecrosslinkerresultsinanunstablefilm,whereasexcessivecrosslinkeralsoresultsinanunstablefilmwherebyexcessiveEGDGEontheelectrodesurfaceresultsinafilmthatdoesnotcompletelydry(EGDGEisaliquidatroomtemperature).SinceEGDGEwillcrosslinkbetweenpolymer-polymer, protein-protein and protein-polymer, the concentration of EGDGE alsodependsontheloadingofproteinperelectrode.FortheimmobilizationofnitrogenaseMoFeproteinwithinapoly(vinylamine)film,anapproximateconcentrationof23mol%

EGDGE to poly(vinylamine) was utilized, resulting in approximately one epoxidefunctionality to every two primary amines present on the poly(vinylamine) backbone(wherea10mgmL-1solutionofpoly(vinylamine)approachessaturation).PreviousworkemployingredoxpolymersandusingEGDGEasthecrosslinkerindicateaconcentrationofbetween20-30mol%EGDGEtobeoptimal.2,3Finally,avolumeof5µLof the finalenzyme/polymer/crosslinker mixture was applied to the surface of a 3 mm glassycarbonelectrode; thisvolumeensure thecompleteyetnon-excessivecoverageof thesurfaceoftheworkingelectrode.Ammonia/ammoniumdetectionassayNH3wasquantifiedaspreviously reported.4,5AnNH3detectionreagentwaspreparedbymixing phosphate buffer (pH 7.3, 0.2M, 100mL), 2-mercaptoethanol (25 µL) andortho-phthalaldehyde (270mg dissolved in 5mL of ethanol) and storing in the dark.Following bioelectrosynthetic experiments, 250 µL of the electrolyte solution(containingNH3producedbyMoFeproteinbioelectrodes)wasmixedwith1000µLoftheNH3detectionreagentandincubatedfor30mininthedark.NH3wasquantifiedbyexcitingthesolutionsat410nmandmeasuringtheiremissionreadingsat472nm.Allsamples were quantified as triplicates and corrected against background samplespreparedwithapo-MoFeproteinbioelectrodes.

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