D Kinetic Isotope Effect of the Hox

Communication pubs.acs.org/JACS

Activation Thermodynamics and H/D Kinetic Isotope Effect of the Hox to HredH+ Transition in [FeFe] Hydrogenase Michael W. Ratzloff,† Molly B. Wilker,‡,§ David W. Mulder,† Carolyn E. Lubner,† Hayden Hamby,‡ Katherine A. Brown,† Gordana Dukovic,‡ and Paul W. King*,† †

Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, United States Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado 80309, United States

‡

S Supporting Information *

ABSTRACT: Molecular complexes between CdSe nanocrystals and Clostridium acetobutylicum [FeFe] hydrogenase I (CaI) enabled light-driven control of electron transfer for spectroscopic detection of redox intermediates during catalytic proton reduction. Here we address the route of electron transfer from CdSe→CaI and activation thermodynamics of the initial step of proton reduction in CaI. The electron paramagnetic spectroscopy of illuminated CdSe:CaI showed how the CaI accessory FeS cluster chain (F-clusters) functions in electron transfer with CdSe. The Hox→HredH+ reduction step measured by Fouriertransform infrared spectroscopy showed an enthalpy of activation of 19 kJ mol−1 and a ∼2.5-fold kinetic isotope effect. Overall, these results support electron injection from CdSe into CaI involving F-clusters, and that the Hox→HredH+ step of catalytic proton reduction in CaI proceeds by a proton-dependent process.

Figure 1. CdSe:CaI complex, and H-cluster reduction scheme. (Left) CdSe:CaI showing photochemically (hv, photon) activated electron (e−) injection from CdSe (green circle) into the CaI (gray) accessory FeS cluster chain (F-clusters) and catalytic site H-cluster (HC, green hexagon). F-clusters include the distal [4Fe−4S] (red square), distal [2Fe−2S] (yellow square), and medial and proximal [4Fe−4S] (brown squares). (Right) Initial reduction step of the oxidized Hcluster, Hox, to form HredH+. Abbreviations; h+, hole; H+, proton; [4Fe−4S]2+, [4Fe−4S]H subcluster; FeIIFeI, FeIFeI, oxidized and reduced [2Fe]H subcluster, respectively.

H

ydrogenases catalyze the reversible activation of H2 at catalytic sites that are composed of low valent, base metals including Ni and Fe. In [FeFe] hydrogenases, the activation of H2 occurs at the H-cluster, which is composed of a [4Fe−4S] cluster ([4Fe−4S]H) linked by a cysteine thiolate to a diiron subcluster, [2Fe]H, with CO, CN−, and azadithiolate (adt) ligands (Figure 1).1−3 Progress on identification of proton-transfer pathways4,5 combined with theoretical Hcluster protonation states6,7 have led to catalytic models6,8,9 that link electron/proton transfer to changes in redox state (Figure S1). The resting state, Hox, is reduced in a series of redox reactions to form H2.6,9,10 Each redox state has a unique CO/CN− ligand infrared absorption (IR) spectrum, and Fourier-transform IR (FTIR) spectroscopy can be used to monitor transitions between these discrete redox states,8,10−15 as well as to follow proton-transfer steps in the catalytic cycle. In this regard, time-resolved FTIR has revealed proton-coupled electron transfer steps in the catalytic mechanism of [NiFe] hydrogenases in unprecedented detail,16,17 however this detail for [FeFe] hydrogenases is lacking. Our previous work has shown that nanocrystals (NC’s) of materials like CdSe, CdTe and CdS have been used to inject electrons into redox enzymes under illumination,18−21 including the [FeFe] hydrogenase I from Clostridium acetobutylicum (CaI) (Figure 1). CaI is a complex enzyme composed of the catalytic H-cluster as well as accessory FeS clusters (F-clusters) © 2017 American Chemical Society

that function in electron transfer. The electrostatic binding of mercaptopropionic acid (MPA) capped NC’s to CaI forms static complexes with molecular orientations that orient the NC’s at the distal F-clusters of CaI (Figure 1).21,22 This orientation supports interfacial electron transfer at kET ≈ 107 s−1,22−24 where CaI catalytic turnover depends on the interval of photoreduction. In this report, we used rapid-scan FTIR (ms scan rate) to measure the formation of HredH+ (Figure 1) in complexes between MPA-CdSe (Figure S2) and CaI (CdSe:CaI) under nonturnover conditions. The temperature and H/D isotope dependence on the observed rate (kobs) of formation of HredH+ were used to determine the thermodynamic properties and solvent kinetic isotope effect (KIE) of the Hox reduction step in CaI, and assess the contribution of protons to the mechanism of reduction. Previous results showed the molecular orientations of MPANC are positioned for electron injection into the distal clusters of CaI (Figure 1). Here we used EPR to assess how electrons enter CaI in CdSe:CaI complexes. The reduction state of CaI was monitored by EPR over time in CdSe:CaI under steadystate illumination (Figure 2). The initial EPR spectrum was Received: April 28, 2017 Published: August 29, 2017 12879

DOI: 10.1021/jacs.7b04216 J. Am. Chem. Soc. 2017, 139, 12879−12882

Communication

Journal of the American Chemical Society

Poisson distribution of CdSe:CaI complexes21 (Table S2) combined with the photoexcitation rate and quantum yield of electron transfer in the absence of a sacrifical donor18,21 (Table S1) result in an electron injection rate from CdSe to CaI of 0.05 s−1, within the time resolution of the FTIR scan rate (ms). Figure 3 shows the time-dependent changes of the 1946 cm−1

Figure 2. EPR spectra of CdSe:CaI complexes under illumination. (A) Oxidized CaI prepared without CdSe showing the Hox signal (g = 2.1, 2.04, 1.99) (x0.5). (B) CaI:CdSe sample prepared in the dark (blue scan), warmed to ∼250 K and illuminated for ∼10−20 s, refrozen in liquid nitrogen, and the EPR spectrum measured at 12 K, and repeated (blue to red). The initial oxidized CaI (blue trace) is enriched in Hox, and electron transfer from CdSe to CaI leads to an attenuation of Hox and accumulation of a complex signal (red trace) of reduced accessory F-clusters. Signal assignments of distal [2Fe−2S]+ and [4Fe−4S]+ clusters are shown based on fits of EPR spectra in Figure S3. Microwave frequency, 9.38 GHz; power, 1.003 mW; temperature, 12 K.

Figure 3. Peak heights (absolute) of νCO bands assigned to Hox (1946 cm−1, top panel) and HredH+ (1899 cm−1, bottom panel). The plots show changes (x-axis, s) in absorption (y-axis, absorbance units, A.U.) at 295 K (H2O, black); 295 K (D2O, orange); 250 K (H2O, green); 200 K (H2O, blue); and 120 K (H2O, red). At 295 K, the IR time resolution is 150 ms per-scan. For all other temperatures, the IR time resolution is 1.5 s per-scan. The t = 0 point is shifted 2.5 s to indicate the onset of illumination.

composed of the Hox H-cluster state, signified by a rhombic g = 2.1 signal and a small fraction of the reduced distal [4Fe−4S] F-cluster (Figure 2A versus 2B). A series of illumination steps led to the gradual formation of a complex, multicomponent signal of reduced F-clusters,25 accompanied by attenuation of the Hox signal indicative of reduction of the H-cluster to the EPR silent, diamagnetic HredH+ state. Detailed analysis of the illuminated CdSe:CaI signal showed intensity assigned to the reduced distal [4Fe−4S] cluster with possible weaker contribution from the reduced [2Fe−2S] cluster (Figure S3 and Figure 2). The EPR results are consistent with our previous findings18,21−24 that electrostatic binding of MPA-capped NC’s to the surface of CaI orients the NC near the distal [4Fe−4S] cluster (Figure 1). To observe the Hox→HredH+ transition in CaI, we followed the changes in the H-cluster IR absorption spectrum of CdSe:CaI under illumination. The Hox IR spectrum arises from a mixed-valent, FeIIFeI, [2Fe]H subcluster coupled to an oxidized [4Fe−4S]H2+ subcluster (Figure S4A). Reduction of Hox forms either a [4Fe−4S]H+−[FeIIFeI], Hred,7,26 or when coupled to proton-transfer forms [4Fe−4S]H2+−[FeIFeI], HredH+ (Figure S4B).7,13−15 Here, we refer to the 1-electron reduced product of Hox as HredH+, where changes in absorbance of the unique vCO bands at 1946 cm−1 (Hox) and 1899 cm−1 (HredH+) were used to determine the kobs for Hox→HredH+ in CdSe:CaI under illumination with 405−410 nm light (Table S1). Samples of CaI were prepared in the Hox state, mixed with CdSe in a 2:1 CdSe:CaI molar ratio, and equilibrated at the reaction temperature to collect the initial IR spectrum. The

(Hox, top panel) and 1899 cm−1 (HredH+, bottom panel) vCO bands of CdSe:CaI under illumination. There is a clear correlation between the decline of Hox and rise of HredH+ vCO bands. As the time of illumination continued at 295 K, further electron injection gradually led to more Hox→HredH+ conversion (Figure S4C). This secondary process further altered the intensity of Hox and HredH+ νCO bands in a nonlinear manner, and a plateau was observed in νCO intensities where the reduction of bound CaI reached saturation (Tables S1 and S2). As temperature decreased from 295 to 200 K, kobs became slower27 relative to the estimated interval of CaI photoreduction (Table S1). This resulted in less change in vCO band intensity (Figure 3), as well as a lengthening of the linear range at lower temperatures (Figure S5). At 120 K, there was no detectable formation of HredH+ in the illuminated CdSe:CaI complexes. Assuming that the steps that precede the Hox→ HredH+ transition are temperature independent, we attribute the temperature-dependence shown in Table 1 to changes in the kinetics of the Hox→HredH+ step. The relative value of kobs for the Hox→HredH+ transition at each temperature was derived from a linear fit of the normalized Hox (kox) and HredH+ (kred) kinetic traces in Figure 3 (Table 1, linear fits from Figure S5). The average of the kox and kred values was used to calculate kobs, for the Hox→HredH+ 12880

DOI: 10.1021/jacs.7b04216 J. Am. Chem. Soc. 2017, 139, 12879−12882

Communication

Journal of the American Chemical Society Table 1. Kinetic Fit Parameters of vCO Traces in Figure 3.a

large (>50) regimes.30 Interfacial electron transfer in CdSemolecular complexes has been shown to have a KIE of ∼1,32 therefore the observation of a KIE can attributed to a dependence of Hox→HredH+ in CaI on proton transfer, assuming that the electron injection step from CdSe is proton-independent. The ratio of kobs(H)/kobs(D) (Table 1) was determined for CdSe:CaI complexes in H2O and D2O buffer at 295 K (Table 1 and Figure S5), and gave a KIE of ∼2.5. This value is within the range of 1.8−2.5 for the KIE of catalytic proton reduction by [FeFe] hydrogenases,31 and together with the temperature dependence of kobs is consistent with the initial reduction step of Hox and formation of HredH+ involving a proton-dependent process in CaI (reaction scheme in Figure 1). In summary, the integration of light-controlled electron transfer with rapid-scan FTIR and steady-state EPR spectroscopies enabled the thermodynamic and H/D isotope dependence of the initial electron reduction step in the catalytic mechanism of [FeFe] hydrogenases. We showed evidence for electrons from CdSe entering CaI by means of the F-cluster chain, which functions both as a conduit and a reservoir of electrons in CaI to facilitate catalytic turnover. We measured the ΔH‡ value of 19 kJ mol−1, and KIE of ∼2.5 for kobs for the Hox→HredH+ step in CaI. Our results are consistent with a proton-dependence for this step in catalytic H2 activation. We have shown that photochemical activation of redox enzymes combined with scanning spectroscopic methods can be used to resolve aspects of catalytic mechanisms, and enable analysis of the thermodynamic landscapes necessary for multielectron reactions in enzymes.

ΔA of vCO bands (s−1)a temp (K)

solvent

295 295 250 200

D2O H2O

Δ1899 cm

−1

(kred)

Δ1946 cm−1 (kox)b

kobsb

0.032 0.067 0.005 0.001

0.022 0.055 0.004 0.001

0.012 0.042 0.004 0.001

a Fits to plots in Figure S5. All SD ± 15% except for kred in D2O with SD ± 45%. bkox are absolute values. kobs = 1/2(kox + kred).

process in the CaI catalytic mechanism (Figure 1). As expected, kobs values decreased, in this case by 10-fold, as temperature decreased from 295 to 200 K. A plot of the kobs values from Table 1 in Figure 4 and fit to the Eyring equation (eq 1) was used to calculate the activation enthalpy (ΔH‡) of Hox→HredH+. ln

kobs k ΔH ‡ ⎛⎜ 1 ⎞⎟ ΔS‡ =− + ln B + T R ⎝T ⎠ h R

(1)

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b04216. Materials and methods, kinetic trace analysis, photoreduction parameters (PDF)

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Figure 4. Eyring plot of CaI νCO kinetics. The temperature and rate constant data from Table 1 (H2O, ●) was fit to eq 1 where

ln

kobs T ‡

( T1 ) − 1.4 (R

= − 2237

2

= 0.92, red dashed lines) to obtain

AUTHOR INFORMATION

Corresponding Author

ΔH = 19 kJ mol−1.

*[email protected] ORCID

The ΔH‡ value of 19 kJ mol−1 that was obtained from the fit (Figure 4) is consistent with gating or conformational barrier for the Hox→HredH+ process in CaI.22 Prior temperaturedependent protein film voltammetry experiments measured ΔH‡ of proton reduction activity by CaI of ∼30 kJ mol−1,28 and QM/MM calculations of proton transfer in CpI, a structural homologue of CaI, predicted values of 38−46 kJ mol−1.5 Neither result measured the ΔH‡ for the discrete 1-electron steps in the catalytic cycle. Moreover, a decline in rate constant of HredH+ formation in CaI at temperatures below 295 K arises from lower activation of proton transfer to drive the Hox→ HredH+ redox transition. This proton-transfer dependence of the initial reduction step in CaI suggests that it is mechanistically related to the proton-coupled electron transfer (PCET) dependent Nia-S to Nia-C step in [NiFe] hydrogenases.29 To further assess the proton dependence of the Hox→HredH+ redox transition, we measured the effect of H2O and D2O (KIE) on kobs at 295 K. The KIE can be used to probe for PCET reactions, where values range over moderate (∼1) to

Carolyn E. Lubner: 0000-0003-1595-4483 Gordana Dukovic: 0000-0001-5102-0958 Paul W. King: 0000-0001-5039-654X Present Address §

Chemistry Department, Luther College, Decorah, IA.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.W.R., D.W.M., C.E.L., K.A.B., and P.W.K. were supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences; and the U.S. Department of Energy under Contract No. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory. Nanocrystal synthesis, characterization, and ligand exchange (M.B.W., H.H., and G.D.) were supported under the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering under Award DE-SC0010334. 12881

DOI: 10.1021/jacs.7b04216 J. Am. Chem. Soc. 2017, 139, 12879−12882

Communication

Journal of the American Chemical Society

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(30) Hammes-Schiffer, S. Acc. Chem. Res. 2009, 42, 1881. (31) Yang, H.; Gandhi, H.; Cornish, A. J.; Moran, J. J.; Kreuzer, H. W.; Ostrom, N. E.; Hegg, E. L. Rapid Commun. Mass Spectrom. 2016, 30, 285−292. (32) Chen, J.; Wu, K.; Rudshteyn, B.; Jia, Y.; Ding, W.; Xie, Z.-X.; Batista, V. S.; Lian, T. J. Am. Chem. Soc. 2016, 138, 884−892.

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DOI: 10.1021/jacs.7b04216 J. Am. Chem. Soc. 2017, 139, 12879−12882