Hydrogenase-Like Electrocatalytic Activation and Inactivation

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Hydrogenase-like electrocatalytic activation and inactivation mechanism by three-dimensional binder-less molecular catalyst Kamal Elouarzaki, Yian Wang, Vishvak Kannan, Haoxiang Xu, Daojian Cheng, Jong-Min Lee, and Adrian C. Fisher ACS Appl. Energy Mater., Just Accepted Manuscript • DOI: 10.1021/acsaem.9b00203 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Applied Energy Materials

Hydrogenase-like electrocatalytic activation and inactivation mechanism by three-dimensional binder-less molecular catalyst Kamal Elouarzaki*+,1,2 Yian Wang+,2,3 Vishvak Kannan,2,4 Haoxiang Xu,5 Daojian Cheng,5,6 Jong-Min Lee* 1, 2 and Adrian C. Fisher*2,3,6 + contributed equally to this work 1 School

of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore 2 Cambridge CARES, CREATE Tower, 1 CREATE Way, Singapore 138602, Singapore 3 Department of Chemical Engineering and Biotechnology, University of Cambridge, West Cambridge Site, Philippa Fawcett Drive, Cambridge CB3 0AS, United Kingdom 4 Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117585 5Laboratory of Molecular and Material Simulation, Beijing University of Chemical Technology, Beijing 100029, P.R. China. 6 International Research Center for Soft Matter, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China KEYWORDS. Molecular catalyst, hydrogenase, heterogeneous catalysis, activation and inactivation mechanism, mathematical model, glucose oxidation, rhodium porphyrin.

ABSTRACT: In response to issues raised by modern energy challenges, molecular electrocatalysis is currently attracting a lot of attention to the tailoring of “model” catalysts, notably understanding the mechanisms, kinetic and thermodynamic parameters that occur during a catalytic reaction. In this regard, nature offers extremely efficient enzymes called hydrogenases. These enzymes that catalyze the reversible interconversions between H2 and H+ at high turnover rates are inactivated by O2. This inactivation yields odd cyclic voltammetric responses originating from a chemical inactivation/redox activation process (IAP). Although IAP has been extensively studied for hydrogenases, their catalytic mechanism is not fully understood because of the intricate but necessary electrical wiring, desorption and complex biochemical environment required. Here, we report a unique example of IAP based on a non-enzymatic catalyst prepared by mixing rhodiumporphyrinic catalyst and an interconnected multi-walled carbon nanotubes matrix which presents an excellent and stable electron transfer. We combined organic synthesis, electrochemistry, mathematical models, and density functional theory calculations to uncover the molecular IAP at the catalytic metallic site. We present a mechanistic analysis of the non-catalytic and catalytic responses exhibited by this complex, enabling a comprehensive understanding of the thermodynamic and kinetic parameters that govern the IAP. These stepwise studies support a mechanism for glucose oxidation that proceeds most-likely through an EC'CE scheme with catalytic steps similar to the ones reported for NiFe hydrogenases. The overall mechanism of the molecular IAP was detailed based on our experimentally validated models and compared to NiFe hydrogenase IAP. Our findings offer novel perspectives to design finely optimized catalysts by eliminating the inactivation phenomena.

INTRODUCTION One significant contemporary challenge for the widespread adoption of energy conversion systems is the need for efficient, stable, and cost-effective catalysts against the efficient but expensive and scarce noble metal catalyst1-3. A very promising perspective in this regard is to tailor an efficient catalyst using

molecular synthetic engineering4-5. This can be achieved by understanding and translating the biological catalytic principles to design “model” synthetic mimics. Delving deep into reaction mechanisms and kinetics and thermodynamic parameters that occur during the catalytic reaction is central to this effort6-10.

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Hydrogenases, remarkable active catalysts for oxidative transformation due to the specific reactive sites of their metal centers, are highly used in this context11-14. This enzyme that catalyses the reversible interconversions between H2 and H + at high turnover rates, is inactivated by O2 converting the active site into different inactive forms. This inactivation dramatically affects the shape of the electrocatalytic response, at the forward scan, the catalytic current due to hydrogen oxidation increases and significantly drops as the potential becomes more positive (i.e., inactivation occurs). However, this behavior is reversed on the subsequent sweep back to a more negative potential (i.e., reactivation occurs). Although the exact mechanism of this inactivation-activation process (IAP) causing this odd shape remains to be elucidated, previous research has provided insights and hypotheses. Limoges and Savéant suggested that the immobilized enzymes on the electrode surface represent a twisted reverse trace behaviour15. The latter is “unusual” in comparison to the “classical” regenerative mechanisms of electrocatalysis16-17. The authors derive a complex electrocatalytic framework involving a five-step reaction scheme to depict the interconversion within the IAP of surface-confined enzymes. This scheme links the hysteresis between forward and backward catalytic responses to the inhibition process. In the mathematical models, the occurrence of twisted reverse oxidation peaks is obtained from a delicate combination of thermodynamic and kinetic parameters of the different reactions involved in the mechanism15. This peculiar behaviour15. This peculiar behavior is better observed when a large rate constant exists for the reactivation of the electrocatalytically active enzyme. Investigations by Armstrong and his co-workers, however, highlighted the experimental mechanistic relationship between these biocatalysts and hydrogen oxidation8, 18-19. They reported a similar behavior to depict the anaerobic interconversion between the inactivation and reactivation of [NiFe]hydrogenase from different microorganisms. A parameter, 𝐸switch derived from the cyclic voltammetry, was defined as the position of the first inflection on the reduction sweep towards lower potential and gives a qualitative diagnosis to compare the ease with which various hydrogenases can be reactivated after oxidative inactivation18. In mechanistic investigations, this parameter was used to infer the acidity constants via pE/pH diagrams, and to calculate the entropies and enthalpies by analysing the dependence on temperature. Nevertheless, Léger et al. suggested that the reduction potential for the socalled NiB state, which is electrocatalytically inactive, cannot be estimated directly by the voltammetric data20. 𝐸switch changes in proportion to the logarithm of scan rate, and for this reason, it was quantified as a kinetic characteristic rather than a thermodynamic parameter20. It was also supported theoretically, by the fact that the normalised governing variables involve the dependence between the 20. It was also supported theoretically by the fact that the normalized governing variables involve the dependence between the IAP’s rate constants and scan rate15..

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electrocatalytic behavior, providing models that feature catalyst IAP. Advantageously, this catalyst presents an excellent and stable electron transfer enabling a straightforward estimation of the thermodynamic and kinetic parameters from its non-catalytic signal. Nonetheless, the NiFe hydrogenase IAP analysis is more challenging because of desorption, electrical wiring, complex chemical environment and instability. Despite these challenges, several works have conducted mechanistic studies to discern and/or attempt to circumvent the IAP in hydrogenase systems22-26. However, we believe this work could eventually be inspirational by testing the robustness of the simulation method adopted for hydrogenase IAP on this molecular-type catalyst. Here, we used a 3D binder-free deuteroporphyrin dimethylester rhodium(III) ((DPDE)RhIII) confined on an interconnected multi-walled carbon nanotube (MWCNT) network to obtain deep insight into the twisted reverse voltammetric responses (Fig. 1) with glucose as a substrate according to the following scheme:

𝑘red ― ⇌ RhI + 2 Rh e 𝑘ox III

𝑘

RhIII + [S] RhI + [P] 𝑘f RhIII + OH ― ⇌ RhIII(OH) 𝑘b 𝑘′red RhIII(OH) + 2e ― ⇌ RhI + OH ― 𝑘′ox Scheme 1. Electrocatalytic scheme of glucose oxidation by (DPDE)RhIII in alkaline medium The study begins with the characterization and mechanistic determination of the non-catalytic reaction scheme (ECE) of the catalyst. Then, the twisted traces were investigated with the introduction of glucose into the system. Based on the experimentally elucidated ECE scheme, we proposed another catalytic EC’CE scheme that is fully consistent with the electrocatalytic data. The mathematical models based on EC’CE enabled the approximation of the reaction kinetics at an elementary level. For better understanding of the key parameters causing this twisted shape, we established the dependence of inactivation and activation reaction rates on potential from experimentally validated EC’CE and generated the 3D surface concentration profiles. We further confirmed the robustness and validity of the EC’CE model with respect to different kinetic conditions (scan rate and concentration). Finally, the overall mechanism explaining the IAP was constructed based on our experimentally validated models and compared to NiFe hydrogenase IAP.

A similar behavior was observed for a bio-derived porphyrin complex modified with rhodium as the catalytic active site. This complex presents an electrocatalytic behavior, which can be consistent with NiFe hydrogenase’s electrocatalytic response21. However, the kinetics and the intricate molecular mechanisms of these interconversions are not fully understood. The extension of this biomimetic capability is subjected to a more detailed thermodynamic and kinetic scrutiny. Mechanistic studies were employed to elucidate the


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ACS Applied Energy Materials with the NiFe hydrogenase were performed in phosphate buffer solution (PBS) at pH = 7.4. pH measurements were conducted during all experiments. All 𝐸1/2 values reported in this work were determined from CV as an average of the oxidative and reductive peak potentials (𝐸ox + 𝐸red)/2. The solutions were deoxygenated by purging and keeping argon prior to each experiment over the solution and during the whole experiment.

SEM and TEM microscopy. The morphology of the modified electrodes was investigated by SEM using an ULTRA 55 FESEM based on the GEMENI FESEM column with beam booster (Nanotechnology Systems Division, Carl Zeiss NTS GmbH, Germany) and a tungsten gun and TEM (JEOL, JEM-2010, 200 kV).

Fabrication

of

𝐌𝐖𝐂𝐍𝐓/(𝐃𝐏𝐃𝐄)𝐑𝐡𝐈𝐈𝐈

electrode.

III

Figure 1. 3D binder-free hybrid (DPDE)RhIII hydrogenase-like electrode (a) Configuration of (DPDE)RhIII molecule (b) Schematic structure of two-electron transfer process from (DPDE)RhIinto (DPDE)RhIIIsupported on MWCNT (c) An example of the “twisted” shape of reverse oxidation trace.

EXPERIMENTAL SECTION Materials. Commercially available analytical grade reagents, α-D-Glucose, Rh2Cl2(CO)4, 1-methyl-2pyrrolidinone (NMP), Deuteroporphine IX dimethyl ester, and potassium hydroxide (KOH) were purchased from Sigma-Aldrich. Commercial grade thin Multi-Walled Carbon Nanotubes (MWCNT) (9.5 nm diameter, purity >98%), obtained from Nanocyl, were used as received without any purification step. Deuteroporphyrine IX dimethyl ester Rhodium(III) ((DPDE)RhIII) was synthetized and characterized by NMR according to literature, electrospray ionization mass spectrometry (ESI-MS), UV-visible spectroscopy21.

Electrochemical Measurements. Cyclic voltammetry (CV) was performed in a conventional three-electrode cell equipped with a glassy carbon (GC) electrode (geometric area = 0.071 cm2), a platinum wire counter-electrode and a saturated calomel electrode (SCE) as reference electrode. The surface of GC electrodes was polished with a 2 µm diamond paste, washed with water using ultrasound, and rinsed with acetone and ethanol. CVs were recorded after three successive reproducible scans. Electrochemical experiments were performed in aqueous KOH solutions of pH = 14, 1 M. Britton-Robinson buffer solutions were used for the preparation of pH = 12. The solution at pH 12 was prepared using Britton_Robinson buffer solutions ([CH3COOH] = 0.1 M, [H3PO4] = 0.1 M, [H3BO4] =0.1 M). The pH of a solution was adjusted by titration using aqueous KOH solutions. The experiments

MWCNT/(DPDE)Rh were prepared with the aim of producing electrodes via -stacking interactions. This MWCNT/(DPDE)RhIII matrix was prepared via a simple filtration of MWCNTs dispersion in presence of (DPDE)RhIII. Experimentally, commercial MWCNTs (5 mg) were dispersed in 10 ml of THF and subjected to sonication for 1 h in order to form a homogeneous suspension. Then, 5 mg of (DPDE)RhIII was added to the MWCNTs dispersion under magnetic stirring. After 12 hours , the mixture was then filtered under vacuum over a PTFE membrane filter with a pore size of 0.45 µm. The formed MWCNT/(DPDE)RhIII matrix was rinsed with THF and deionized water to remove any unbound components. The formed powder on a PTFE membrane was then dried in a vacuum oven at 80 ℃ for 60 mins and carefully peeled off the surface of the filter. The MWCNT/(DPDE)RhIII electrode was fabricated by adding MWCNT/(DPDE)RhIII powder to 1 ml of NMP solvent. The suspension was homogenized by magnetic stirring for 60 mins. The resulting supernatant was used as catalyst ink. For electrochemical investigations, a portion (20 µL) of this suspension was dropped onto a freshly polished GC disk electrode (3 mm diameter) and the solvent was removed under reduced pressure.

Fabrication of 𝐌𝐖𝐂𝐍𝐓/𝐍𝐢𝐅𝐞 Hydrogenase electrode. Aquifex aeolicus [NiFe]-hydrogenase was purified and prepared as described27. The deposition27. The deposition of MWCNT was performed by drop-coating of 20 µL of MWCNT dispersion in NMP on GC electrodes followed by evaporation of the solvent under vacuum. The modified electrodes were then washed with phosphate buffer solution (PBS) several times. Finally, the electrodes were incubated in 20 µL of [NiFe]-hydrogenase (5 µM in PBS pH = 7.4) for 4 hours at 4 ℃. The resulting electrodes were then washed with phosphate buffer solution.



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Figure 2. Characterization of the electrode material. Deconvoluted cyclic voltammograms for the non-catalytic (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼𝐼𝐼 at different scan rates 𝜈 = 5, 10, 20, 50, 75, 100, 150, 200, 500 𝑚𝑉/𝑠 in 1 M aq. KOH at pH 14(A). SEM image of the carbon nanotubes modified with (𝐷𝑃𝐷𝐸)𝑅ℎ (B(i)), TEM image of the carbon nanotube before modification (B(ii)) and TEM image of the carbon nanotube modified with (𝐷𝑃𝐷𝐸)𝑅ℎ (B(iii)).

RESULTS AND DISCUSSION General non-catalytic electrochemical properties of surface confined (𝐃𝐏𝐃𝐄)𝐑𝐡𝐈𝐈𝐈. The (DPDE)RhIII was immobilized onto MWCNT by mixing (DPDE)RhIII and MWCNTs in THF under magnetic stirring. After 12 hours, the mixture was filtered under vacuum over a PTFE membrane. The binder-free (DPDE)RhIII/MWCNT matrix was rinsed with THF and deionized water to remove any unbound components. The resulting dried powder was peeled off from the surface of the filter and re-dispersed in THF. The morphological changes and the nanoscale mixture proprieties of (DPDE)RhIII/MWCNT matrix were characterized both by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). To carry out TEM, drops of dilute (DPDE)RhIII/MWCNTs matrix in THF were deposited onto electron microscopy grids and allowed to dry. TEM revealed that the immobilized porphyrins are distinguished at the sidewalls of the MWCNTs compared to the MWCNTs in absence of porphyrins Fig. (2B (ii-iii)). It clearly appears that the layer formation increases the diameter of the nanotubes from an average diameter value of 8-10 nm to 12-16 nm. Finally, the 3D binder-free (DPDE) RhIII/MWCNT electrode was prepared by dispersing (DPDE)RhIII/MWCNT in NMP. The resulting homogenous dispersion was placed on top of the glassy carbon (GC) and evaporated, leaving a stable film. Based on this transfer process, a reproducible 15 ― 20 µm thick film was obtained on the electrodes. SEM images (Fig. 2B(i)) confirmed that the MWCNT film exhibits a 3D porous high-branched interconnected MWCNT network nanostructure, indicative of the high electroactive area of the electrodes. The hybrid (DPDE)RhIII film onto GC was directly used as electrodes to which no polymer binder or additives were added. A series of successive deconvoluted28 cyclic

voltammograms (CVs) observed when the potential is scanned between 0 and ―1.2 V at pH = 14 are presented in Fig. (2A). The CVs display a well-defined reversible peak at ―485 V vs. SCE assigned to the Rh redox couple of the complex (DPDE)RhIII. As the scan rate is systematically varied from 0.005 to 0.5 V/s, the analysis of the electrochemical characteristics, including E–ν plot of the peak positions in relation to the scan rate where υ is the scan rate, indicates a two-electron surface-confined process with a coverage of 3.61 × 1014 molecules cm-2 (Supplementary Figure 1). Furthermore, upon repetitive sweeping over periods up to 5 h, the peak heights change by less than 3%, indicating that these electrodes are remarkably stable over time in alkaline conditions.

Non-catalytic electrochemical mechanism of surface-confined (𝐃𝐏𝐃𝐄)𝐑𝐡𝐈𝐈𝐈. To delve deep into the overall mechanism, digital simulations of the recorded CVs were used. To achieve this, the 𝐸01,2,3 values, the standard heterogeneous rate constants (𝑘1,2,3 ), the chemical rate 0 constants (𝑘f/𝑘b), and 𝛼 must be estimated. These values will then be used in a semi-empirical Butler-Volmer29 (BV) formalism including the effect of the rate constant to obtain the model predictions, as 𝐼 = 𝐹𝐴

3

𝐼𝑗

∑ 𝐹𝐴 = ― 𝑘

𝑗 red𝛤ox

+ 𝑘𝑗ox𝛤red

(4)

𝑗=1

where the rate constants of BV model, 𝑘𝑗red and 𝑘𝑗ox, have an exponential dependence with the applied potential which can be written as, 𝑘𝑗red = 𝑘𝑗0𝑒 𝑘𝑗ox

=

𝑘𝑗0𝑒

𝐹 ―𝛼 (𝐸 ― 𝐸0𝑗 ) 𝑅𝑇

(1 ― 𝛼)


𝐹 𝑅𝑇

(𝐸 ― 𝐸0𝑗 )

(5) (6)

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The definitions of all the symbols are summarized in Supplementary Table 1. We propose an ECE scheme to represent the non-catalytic electrochemical behavior of (DPDE)RhIII as follows:

𝐸

𝐶

𝑘10 𝑘20 ― II (1) (DPDE)Rh + e ⇌ (DPDE)Rh + e ⇌ (DPDE) RhI 𝐸01 𝐸02 𝑘f (2) (DPDE)RhIII + OH ― ⇌ (DPDE)RhIII(OH) 𝑘b III

―

(DPDE)RhIII(OH) + e ― 𝐸 +e

―

𝑘30 ⇌ (DPDE)RhII +OH ― 𝐸03

𝑘20 ⇌ (DPDE)RhI+ OH ― 𝐸02

(3)

Scheme 2. ECE mechanism followed by (DPDE)Rh in the absence of a substrate in alkaline conditions

The mathematical model was constructed based on Scheme 1 with the BV formalism as the boundary condition for each species (Supplementary Method 2). The apparent heterogeneous rate constant k0 and 𝛼 were estimated from Laviron’s formalism30 as 2.5 ± 1.5 s ―1 and 0.48 ± 0.01 respectively. k0 was calculated for a 𝛥𝐸 of 121 mV at 500 mV/s and 𝛼 was calculated from the slopes of the curves in Supplementary Figure 2. The validity of Laviron’s formalism is confirmed by the halfway peak width of 60 mV, which is close to the theoretical value of (62.5/𝛼𝑛) mV 30. This calculated k0 is relatively slow30. This calculated k0is relatively slow in that it provokes a peak splitting at higher scan rates31. An increased ∆𝐸 at these scan rates is most likely observed when the time constant of interfacial kinetic is close to the imposed time scale of the voltammetric sweep, 𝑅𝑇/𝐹𝜐. To consolidate this experimental value, we carried out density function theory (DFT) calculations of the rhodium active site (Supplementary Method 1) to compute the energy difference between the (DPDE)RhI and (DPDE) RhIII coordinated to hydroxide via two one-electron transfers. The estimated interfacial transfer rate constant of 𝑘0 = 2.16 s-1 agrees well with the experimental value. In addition, at pH = 14, the 𝐸0 values for the electrochemical redox couple of the Rh complex was estimated from the experimental CVs and the relation between the chemical coordination rate constant is obtained from the pKa of the equilibrium between RhIII(OH) and RhIII 21.

Figure 3. Validation of the model predictions for the noncatalytic (DPDE)RhIIIExperimental validation of the simulated CV curve at 𝜈 = 150 mV/s and in 1 M aq. KOH at pH = 14 (A) and at 𝜈 = 50 mV/s and in 1 M aq. KOH at pH = 12 (B), assuming Butler-Volmer kinetics and Laviron’s model. The solid lines (─) represent experiments and the blue circles (○) represent the model predictions.

Simulations were calibrated against the experimental data following the conditions and parameters in Supplementary Method 2. Figure (3A) shows the calibration of the simulated CV with use of the values listed in Table 1. The error in fitting with the estimated set of parameters was quantified for each case by the root mean square (RMS) between the experiments and the model predictions. For example, the RMS value for Figure (3A) was 2.5 × 10 ―5. The

best set of parameters with the lowest RMS values, show a good correspondence between the simulated and experimental CV. Moreover, the experimental validation of the model for different scan rates is also presented in Supplementary Figure 3. The simulations were able to accurately describe the trend of the experimental CVs produced at pH 14 with different scan rates. The agreement with experiments suggests that the mechanism of electron transfer most-likely follows the proposed ECE-type mechanism with a chemical reaction coupled with two electron-transfer reactions. According to this mechanism, the reduction wave observed on the forward scan involves two one-electron reductions separated by a chemical step. In strong alkaline conditions, the chemical step is a hydroxide coordination step producing RhIII(OH) from RhIII 21, 32. The reverse scan shows one wave corresponding to two one-electron oxidations where the reduction of RhIII(OH) to RhI is thermodynamically more favourable than that of RhIII at pH = 14 (𝑘f ≫ 𝑘b and pKaRhIII(OH)|RhIII = 6.4)21, 33. In addition, the ECE scheme presented above, the RhII intermediate formed is the rate limiting step (𝑘20 < 𝑘10 = 𝑘30) 21 indicating that the lifetime of the RhII is extremely short 2 to be captured at this pH. Therefore, 𝑘app 0 ≃ 𝑘0 which is the rate constant of the formation of the RhII intermediate. Furthermore, as 𝑘f ≫ 𝑘b at pH 14, the Scheme 2 can simply be treated as an EE mechanism with a C branch, during the estimation of the electrochemical parameters. To ensure further validity of this reaction path, the system was interrogated at a different thermodynamic condition by decreasing the pH value (pH = 12). Under this condition, the CV shows that the two one-electron transfer process becomes distinguishable compared to pH 14. Fig 3B. shows the experimental validation of this difference in the responses at pH 14 and pH 12 captured by the mathematical model, which supports the steps presented in Scheme 2. All the numerical analyses were performed using a finite difference solver MATLAB34 and the parameters used in the mathematical models are listed in Supplementary Table 2. Table 1. Parameters for calibration of the non-catalytic CVs 𝐾𝐴 = 𝑘f/𝑘b 𝑘20 /s ―1 𝛼 3.5


107

0.5


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Scheme 3. EC′CE reaction mechanism of the IAP at pH = 14.

Identification of the IAPs. After investigating the characteristics of MWCNT/(DPDE)RhIII electrodes, glucose was introduced as a substrate into the system. Experimental response at 10 mV/s can be observed in Figure 4A. Upon addition of glucose, the current due to glucose oxidation increases, and begins to level off as the potential becomes more positive to reach a maximum value close to𝐸1/2 (ca. ―0.485 V). As the potential is raised above 𝐸1/2, the current drops dramatically due to the inactivation mechanism. However, this inactivation is reversed by a reactivation on the subsequent sweep back to a more negative potential. Similar trends of CVs were obtained with NiFe hydrogenase/MWCNT/GC with an NiFe active site (Fig. (4A(inset)) as previously reported for an enzymatic IAP11-14, 18, 20. To analyze the mechanisms for the occurrence of IAP, we adopt a new scheme derived from the combination of the aforementioned ECE scheme 1 and Limoges and Savéant’s enzymatic catalytic scheme15. The mechanism is hypothesized as an EC′CE scheme which connects the active and inactive forms according to electrochemical terminology29. At pH 14, the electrochemical steps are simplified to involve simultaneous two electron transfer processes during the catalytic cycle. The form of this EC′CE reaction is summarised by equations (7) - (10), 𝐸 𝐶′ 𝐶

𝑘red [(DPDE)Rh ] + 2e ⇌ [(DPDE)RhI] 𝑘ox ―

III

𝑘

𝐸

(8)

[(DPDE)RhIII] + [S] [(DPDE)RhI] + [P] 𝑘f [(DPDE)RhIII] + OH ― ⇌ [(DPDE)RhIII](OH) 𝑘b 𝑘′red

(7)

(9)

― [(DPDE)RhIII](OH) + 2e ― ⇌ [(DPDE)RhI] + OH(10)

𝑘′ox

𝑘red, 𝑘ox, 𝑘′red, and 𝑘′ox are the rate constants of the electrochemical reactions and 𝑘, 𝑘f and 𝑘b are the rate constants of the chemical reactions. The surface reaction is suggested to follow the empirical BV formalism for electron transfer and the diffusion of glucose obeys Fick’s law in the solution. The robustness and validity of Scheme 3 was evaluated by mathematical models. The equations for the model used in the EC′CE simulations are derived as follows. The governing equations for the surface concentrations of the rhodium species are: ∂𝛤RhI 𝐼RhIII/RhI 𝐼RhIII(OH)/RhI (11 =― + 𝑘[S]𝛤RhIII ― ) ∂𝑡 𝑛𝐹𝐴 𝑛𝐹𝐴 ∂𝛤RhIII 𝐼RhIII/RhI (12 = ― 𝑘[S]𝛤RhIII ― 𝑘f𝛤RhIII + 𝑘b𝛤RhIII(OH) ) 𝑑𝑡 𝑛𝐹𝐴 ∂𝛤RhIII(OH) 𝐼RhIII(OH)/RhI (13 = 𝑘f𝛤RhIII ― 𝑘b𝛤RhIII(OH) + ) ∂𝑡 𝑛𝐹𝐴 The total surface concentration is constant and can be given as 𝛤RhI + 𝛤RhIII + 𝛤RhIII(OH) = 𝛤 ∗ (14) Substrate transport to the electrode is given by the linear diffusion equation as ∂[S] ∂2[S] (15) = 𝐷s 2 ― 𝑘[S]𝛤RhIII ∂𝑡 ∂𝑥 The contributions to the measured current are given as 𝐼RhIII/RhI and 𝐼RhIII(OH)/RhI corresponding to the oxidation of (DPDE)RhI to (DPDE)RhIII and the reduction of (DPDE)RhIII (OH) to (DPDE)RhI, respectively. The total current for an applied potential is given as,

Figure 4. Validation of the model predictions for the electrocatalysis of (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼𝐼𝐼 and the kinetic parameters as a function of the applied potential. Inset: Voltammogram of hydrogenase Experimental validation of the model predictions showing the electrochemical reactivation of (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼𝐼𝐼 catalyst in the presence of 50 𝑚𝑀 glucose in 1 M aq. KOH at 𝑝𝐻 = 14 and at 𝜈 = 10 𝑚𝑉 𝑠 ―1 (A). Voltammogram of hydrogenase with an NiFe active site at 45 oC in PBS at 𝑝𝐻 = 7.4 under 𝐻2 and at 𝜈 = 5 𝑚𝑉 𝑠 ―1 is presented in the inset (A). Kinetic parameters 𝑘′𝑜𝑥 = inactivation rate (blue line), 𝑘′𝑟𝑒𝑑 = reactivation rate (red line) and 𝑘[𝑆] = catalytic rate at a substrate concentration of 50 𝑚𝑀, as a function of the applied potential (𝐸) (B).


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20.20. This attributes to the reductive [ ― 𝑘′red𝛤RhIIIreported 𝐼 = 𝐼RhIII/RhI + 𝐼RhIII(OH)/RhI = [ ― 𝑘red𝛤RhIII + 𝑘ox𝛤RhI] + (16) earlier (OH) + 𝑘′ox𝛤 RhI] oxidation peak being quasi-symmetrical to the oxidation peak in Fig. (4A). From the electron transfer rate constant 𝑘0 obtained in the During the forward scan, 𝑘′ox (inactivation rate) increases previous section, the rate constants for the reactivation step exponentially when the potential increases and dominates can be given following the BV model as the process under very oxidizing conditions Fig. (4B) in 𝐹 ―𝛼 (𝐸 ― 𝐸0RhIII(OH)/RhI) sharp contrast with the result collected for NiFe (17) 𝑅𝑇 ′ = 𝑘0′ 𝑒 𝑘red hydrogenase where the rate of inactivation is independent 𝐹 of potential 20.20. This difference in terms of rate of (1 ― 𝛼) (𝐸 ― 𝐸0RhIII(OH)/RhI) (18) 𝑅𝑇 𝑘′ox = 𝑘′0𝑒 inactivation engenders a sharper decreasing slope on the forward scan for the present catalyst molecular catalyst. with the electron transfer rate constant for equation (10), Mechanistically, by increasing the potential, the 𝑘′0 is assumed to be proportional to 𝑘0, as both are electron catalytically active form (DPDE)RhI is oxidised to (DPDE) transfer limited reactions, according to the relation RhIII Fig. (5A-B). The chemical inactivation reaction can be 𝑘0 = Φ𝑘′0 (19) considered as quasi-irreversible because the rate of the The standard potentials of both the electrochemical chemical coordination step, 𝑘f has a value 107 times higher reactions in equations (7) and (10) are assumed to be equal than 𝑘b(Table 2). This suggests that the coordination of the i.e. 𝐸0RhIII/RhI = 𝐸0RhIII(OH)/RhI (Fig 3A)21.21. Hence, parameter generated (DPDE)RhIII and formation of chemically inactive estimation was carried out using the experimental data for (DPDE)RhIII(OH) is the dominating process at high different values of Φ to obtain 𝑘0, 𝑘, 𝑘f, 𝑘b, and 𝑘′0 found in potential yielding a high surface concentration of (DPDE) Supplementary Table 3, and the initial and boundary RhIII(OH) after attaining 0.2V as seen in in the Nernst plot in conditions for the governing equations (11) - (13) and (15) Fig. (5C) compared to Fig. (5A-B). The catalytic current are given in the Supplementary Method 3. To the best of our dramatically dropping to almost 0 A is a consequence of this knowledge, obtaining a closed form analytical solution for non-regeneration of (DPDE)RhIII. The fact that the rate of the system of partial differential equations (11) - (13) and activation tends to zero at high potential confirms that the (15) is not straightforward. Therefore, a numerical reactivation process is only driven by an electron transfer integration procedure is adopted and is implemented using from the electrode. 35a finite difference solver to obtain the model predictions 37.

The validation of the model for 150 mM at 10 mV/s is presented and for the other set of experiments performed at different scan rates in Fig. (4A) and Supplementary Figure 4 and 5, respectively. The agreement between the calculated and experimental CVs is good over a range of scan rates and the estimated physical parameters obtained from this model are given in Table 2.

Table 2. Parameters estimated from the voltammograms showing the catalytic behavior of (𝐃𝐏𝐃𝐄)𝐑𝐡𝐈𝐈𝐈 𝐾𝐴 = 𝑘f/𝑘b 𝑘0/s ―1 𝑘/cm3 s ―1 mol ―1 Φ 3.5

(1 ± 0.1) × 105

107

1 ± 0.1

Before discussing the mechanistic implications of scan rates and concentrations, we first explain how a subtle combination of thermodynamics and kinetics of the reactions results in the twisted reverse CV. This can be obtained by analyzing the dependence of 𝑘′red (reactivation rate) and 𝑘′ox (inactivation rate) on potential. For this reason, we have applied the validated EC′CE model to generate the profiles of 𝑘′red and 𝑘′ox as well as the 3D surface concentration profiles of all the electroactive species at a given potential range which is important to indicate the limiting process and predict the pathway that the system follows. The generated curves are presented in Fig. (4B) and (5). From Fig. (4B), the rate of activation and inactivation are symmetrical, and both are dependent on the potential unlike the rate constants of a few enzymatic systems



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Figure 5. Nernst plots. Nernst plot relating the normalised concentration of (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼 with the inactivation rate constant 𝑘′𝑜𝑥 (A) and reactivation rate constant 𝑘′𝑟𝑒𝑑 (D) and the applied potential; Nernst plot relating the normalised concentration of (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼𝐼𝐼 with the inactivation rate constant 𝑘′𝑜𝑥 (B) and reactivation rate constant 𝑘′𝑟𝑒𝑑 (E) and the applied potential; and Nernst plot relating the normalised concentration of (𝐷𝑃𝐷𝐸)𝑅ℎ𝐼𝐼𝐼(𝑂𝐻) with the inactivation rate constant 𝑘′𝑜𝑥 (C) and reactivation rate constant 𝑘′𝑟𝑒𝑑 (F) and the applied potential. The red lines illustrate the inactivation/ reactivation rate constants as a function of the applied potential.

In contrast, 𝑘′red (activation rate) increases exponentially when the potential decreases and governs the process at very negative potential. Similar dependence has been observed for NiFe hydrogenase20.20. As the backward scan commences, the large generated surface concentration of (DPDE)RhIII(OH) Fig. (5F) gets electrochemically reduced into (DPDE)RhI intermediate (ready) when the potential approaches 𝐸1 2 . (DPDE)RhI is referred to as an intermediate here, as it gets oxidised spontaneously to form (DPDE)RhIII owing to the high rate constant 𝑘′𝑜𝑥 at the operating potential (𝐸 ≥ 𝐸1 2 = ― 0.485 V) in Fig. (4). During this oxidation process, there exists a competition between the coordination of glucose and the coordination of hydroxide. However, the surface concentration of (DPDE) RhIII(OH) ≫ (DPDE)RhIII(glucose) (Fig. (5E) and (5F)) in this potential range and thus, the formation of (DPDE)RhIII (glucose) is not thermodynamically favored until the potential approaches 𝐸1/2. An increase in the concentration of (DPDE)RhIII in Fig. (5E) explains the appearance of the reductive reactivation catalytic peak due to the catalytic oxidation of glucose, as seen in Fig. (3). Moreover, from Fig. (4), the rate of activation and inactivation are symmetrical, and both are dependent on the potential unlike the rate constants of a few enzymatic systems reported earlier20.20.

data at different operating conditions. From Supplementary Figure 4, it can be observed that the shape of the CVs is very sensitive to scan rates. Thus, the contribution of each reaction varies according to its kinetic parameters. To add depth to the analysis, the effect of scan rate was taken into account for the model predictions and the validation is provided in Supplementary Figure 5. One immediate inference is that as the scan rate decreases, the reactivation (twisted peak) becomes more apparent. In contrast, at high scan rate a reductive peak appears. With further increase in scan rate, the reduction peak becomes more dominant. Two factors could lead to such behavior: First, the rate of the catalytic chemical reaction, which is a second order reaction. For a better use of this factor, this reaction should be converted to a first order reaction by multiplication with the concentration (reaction turnover) and its reciprocal equates the time constant of the catalytic reaction as 𝑡c = 1/𝑘[𝑆]. The second factor is the natural time scale of voltammetry given as 𝑡v = 𝑅𝑇/𝐹𝜈. By comparing these two factors, which measure the interference of the inactivation reaction against the timescale of the experiment governed by the scan rate, the change of the CV shapes can be explained based on the validated EC′CE mechanism.

Mechanistic analysis of kinetics of the reactions. After investigating and expounding the mechanism leading to the odd shape in the previous section, we now examine whether the EC′CE model is able to reproduce the available kinetic


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ACS Applied Energy Materials In addition, it should be noted that, when 𝑡c for Fig. (6B) was decreased through forced convection by a rotating disk electrode (RDE) at 2500 rpm ([S] and 𝜈 unchanged), the reduction peak disappears. Compared to the stagnant medium, the convective flux at the RDE increases the catalytic turnover which in turn decreases the 𝑡c. These observations further bolster the above reasoning.

Figure 6. Validation of the model predictions of (DPDE)RhIII for different 𝜆. Experimental validation of the model predictions of the catalytic behavior of (DPDE)RhIII at 𝑣 = 50 mV/s for 𝜆 = 𝑘[S]/𝜈 at [S] = 0 mM (A),50 mM (B), 150 mM (C) and 225 mM (D) in 1 M aq. KOH at pH = 14.). The solid lines (─) represent experiments and the blue circles (○) represent the model predictions.

The 𝑡c is 0.2 s for a glucose concentration of 50 mM (Supplementary Figure 5). For 𝜈 = 10 mV/s, 𝑡v ≫ 𝑡cand this condition yields a twisted peak in the reductive scan and no reduction peak corresponding to the reduction of (DPDE)RhIII(OH) can be obtained (Supplementary Figure 5A-B). In this case, the catalytic reaction is fast relative to the time scale of the experiment. As 𝑡c approaches 𝑡v, a distinguishable reduction peak is obtained in addition to the twisted peak (at 𝜈 = 100 mV/s) (Supplementary Figure 5CD). Here, the chemical reaction is in the same range with respect to the time scale of the experiment. However, at 𝜈 ≥ 200 mV/s, 𝑡v ≪ 𝑡c and the electroreduction (DPDE) RhIII(OH) to (DPDE) RhI is dominating the process and theoretically, as ν →∞, the reduction peak becomes purely reversible. This condition explains that the electrocataysis does not proceed under the fast scan rate because of the slow catalytic turnover and the rapid electroreduction by two-electrons of any formed (DPDE)RhIII(OH) quenches the catalytic reaction and thus the whole cycle. In other words, the reactivation period is shortened at a relatively fast scan rate or even absent at very fast scan rate. Therefore, the twisted peak corresponds to the entire catalytic cycle and the reduction peak corresponds to the electrochemical regeneration of (DPDE)RhI from (DPDE)RhIII(OH) by the electrode. This behaviour is in consensus with the effect of the kinetic parameter, 𝜆 = 𝑘[S] 𝑣 discussed by Savéant et al.15. We expect that the above reasoning should not fail when 𝑡c changes by changing substrate concentrations [S] at a fixed experiment time scale. To check this, we performed experiments at different [S]. Fig. (6) presents the resulting CVs as well as their validations for different values of 𝑡c. At small 𝑡c values ([S] large, high turnover), the contribution of the catalytic rate constant engenders the twisted peak behaviour in Fig. (6C-D). In contrast, for a large 𝑡c value ([S] small, low turnover), the electroreduction peak is obtained.

Figure 7. 𝐸switch Inset: 𝐸switch for NiFe hydrogenase catalysis. Derivative of the current/potential profile during the reductive scan of the voltammogram obtained for the catalytic behavior of (DPDE)RhIII in 1 M aq. KOH at pH =14 in the presence of 150 mM glucose at 𝜐 = 20 mV/s (black line), 50 mV/s (blue line), 75 mV/s (brown line), 100 mV/s (green line), 150 mV/s (purple line) and 200 mV/s (red line). The inset presents the derivative of the current/potential profile during the reductive scan of the voltammogram obtained for the catalytic behaviour of hydrogenase with an NiFe active site in PBS at pH = 7.4 at 45 oC under H2 at 𝑣 = 5 mV/s.

Mechanistic analysis of kinetics using hydrogenase parameter 𝑬switch. Considering that 𝐸switch is extensively

used in enzymatic research to characterize hydrogenases, the analysis of the molecular IAP with respect to 𝐸switch was studied in a similar way to that of hydrogenase IAPs. 𝐸switch was introduced by Armstrong et al. and is a potential value at which the enzyme undergoes reductive reactivation with a switch on potential18. The switch potential can be calculated from the first derivative 𝑑𝐼/𝑑𝐸 of the CV, where the minimum corresponds to the 𝐸switch potential. As noted before, the experiments recorded successively with the same (DPDE)RhIII electrode at 6 different scan rates (Supplementary Figure 4) show that the shape of the derivative 𝑑𝐼/𝑑𝐸 curves is strongly dependent on scan rate (Fig. (7)). An 𝐸switch at ―0.367 V can be observed corresponding to the reductive interconversion between the inactive and active form. The profile of the 𝑑𝐼/𝑑𝐸 curve showing 𝐸switch is strikingly similar to the 𝑑𝐼/𝑑𝐸 curve of NiFe hydrogenase with an NiFe active site at 45 °C in pH = 7.4 under H2 (Fig. (7 inset)). It is noteworthy that the 𝐸′switch at ―0.525 V appears at 𝜈 ≥ 100 mV/s and gets more pronounced and dominant as the scan rate increases, while the 𝐸switch at ―0.367 V significantly decreases and disappears at 200 mV/s. Additionally, at 𝑣 = 10 mV/s, 𝑡v ≫ 𝑡c yields only one 𝐸switch. 𝐸switch is actually the potential at



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Figure 8. Overall catalytic cycle. Inset : Proposed overall catalytic cycle of hydrogenase with an Ni active site.

which the catalytic turnover is inferior to the reciprocal of the natural time constant of the experiment. As 𝑡c → 𝑡v , both the 𝐸switch and 𝐸′switch coexist (at 𝜈 = 100 mV/s). During this transition state, the value of 𝐸switch is slightly shifting from the fact that another 𝐸switch is appearing. This may be key to explain the dependence of the 𝐸switch value on scan rate and the effect of the electrical wiring of hydrogenase. 20 At 𝑡v ≪ 𝑡c, the catalytic reaction cannot be captured and only 𝐸′switch corresponding to the regeneration of (DPDE)RhI can be obtained. 𝐸′switch is the potential at which the catalytic turnover is greater than the reciprocal of the natural time constant of the CV. Therefore, 𝐸switch corresponds to the entire catalytic cycle and 𝐸′switch corresponds to the electrochemical regeneration of (DPDE)RhI from (DPDE) RhIII(OH). Another proof supporting the switch mechanism is the occurrence of an isopotential point38-39 (IPP) at ―0.477 V in Fig. (7). An IPP, the electrochemical equivalent to an isosbestic point in spectroscopy, arises in voltammetry when one electroactive species undergoes a transformation to another. The existence of an IPP implies that the transformation is quantitative, and the sum of the reactants and products remain constant. If there are any side reactions involving either set of species, an IPP will not be ensured. Therefore, the IPP in Fig. (7) is attributed to the transition between 𝐸switch and 𝐸′switch. IPP is observed for this molecular catalyst, but, to the best of our knowledge never for a system where NiFe hydrogenase is immobilized. This is consistent with the fact that electronic connection of the hydrogenase active site to the electrode is very tricky compared to the present molecular catalyst.

Our results conclude that 𝐸switch is not the reduction potential of the inactive or active form at different operating potentials but it strongly depends on the reduction potential of the inactive form and/or reaction(s) responsible for the reactivation process. Experimentally, this dependence is related to the quality of the electrical connection of the inactive and active site to the electrode, the profile of the rate constant of reactivation and the imposed time scale of the CV. Following these results, the proposed catalytic mechanism for the overall IAP is presented in Fig.8 in a way similar to that of a NiFe hydrogenase catalytic cycle (Fig.8 inset) 40.

CONCLUSIONS The present study elucidates the mechanism of molecular IAP (summarized in Fig. (8)) and shows how the kinetic and thermodynamic parameters of the surface-confined rhodium-porphyrinic catalyst causes odd voltammetric traces. The combination of experimental results and mathematical models confirms that the role of the hydroxide ions as an inhibitor in the catalytic cycle is comparable to the inactivation process of NiFe hydrogenase during the hydrogen oxidation. However, hydroxide ions play a significant role in the glucose deprotonation to facilitate the catalytic reaction at the metal center. Such a deprotonation step can be replaced by the introduction of Brønsted bases avoiding chemically inactive and unfavorable intermediates that arise from the hydroxide coordination. Thus, Brønsted bases may be employed to test the similarities of the inactivation of the porphyrins at pH 7


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and compare it with hydrogenase at this pH, which could lead to important breakthroughs in electrocatalysis. In addition, two-electron oxidation steps are intrinsic to electrochemical reactions involving H2, CO2 and numerous other molecules. These results and observations might have important impacts and implications for the development of efficient molecular electrocatalysts composed of very low loading of precious metals, with applications extending beyond alcohol or sugar oxidation.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected]

Author Contributions K. E., A. F. and J.-M. L. designed the performed experiments. K.E. was responsible for the organic synthesis. K. E. and A. F. designed the electrochemical characterization. K. E. conducted all experiments. Y. W., V. K., A. F. and K. E. developed mathematical models and the electrochemical result analysis. D. C. and H. X. were responsible for DFT calculations. J.-M. L. was responsible for the project management. K. E, V. K., Y. W. and J.-M. L. prepared the manuscript.

Funding Sources This project is funded by the National Research Foundation (NRF), Prime Minister's Office, Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.

REFERENCES (1) Yan, Y.; Xia, B. Y.; Ge, X.; Liu, Z.; Fisher, A.; Wang, X. A flexible electrode based on iron phosphide nanotubes for overall water splitting. Chemistry-A European Journal 2015, 21, 1806218067. (2) Zhang, J.; Zhao, Z.; Xia, Z.; Dai, L. A metal-free bifunctional electrocatalyst for oxygen reduction and oxygen evolution reactions. Nature nanotechnology 2015, 10, 444-452. (3) Zeng, Z.; Chang, K.-C.; Kubal, J.; Markovic, N. M.; Greeley, J. Stabilization of ultrathin (hydroxy) oxide films on transition metal substrates for electrochemical energy conversion. Nature Energy 2017, 2, 17070. (4) Elouarzaki, K.; Mandoc, L.-R. P.; Gorgy, K.; Holzinger, M.; Amarandei, C.-A.; Ungureanu, E.-M.; Cosnier, S. Synthesis and electrochemical characterization of original “TEMPO” functionalized multiwall carbon nanotube materials: Application to iron (II) detection. Electrochemistry Communications 2015, 60, 131-134, DOI: https://doi.org/10.1016/j.elecom.2015.08.024. (5) Altamura, L.; Horvath, C.; Rengaraj, S.; Rongier, A.; Elouarzaki, K.; Gondran, C.; Maçon, A. L.; Vendrely, C.; Bouchiat, V.; Fontecave, M. A synthetic redox biofilm made from metalloprotein–prion domain chimera nanowires. Nature chemistry 2017, 9 , 157. (6) Nekrassova, O.; Allen, G. D.; Lawrence, N. S.; Jiang, L.; Jones, T. G.; Compton, R. G. The oxidation of cysteine by aqueous ferricyanide: a kinetic study using boron doped diamond electrode voltammetry. Electroanalysis 2002, 14, 1464-1469. (7) Deshpande, A. G.; Gu, Y.; Matthews, S. M.; Yunus, K.; Slater, N. K.; Brennan, C. M.; Fisher, A. C. Hydrodynamic focusing studies in microreactors using voltammetric analysis: Theory and experiment. Chemical Engineering Journal 2009, 149, 428-434.

(8) Hexter, S. V.; Chung, M.-W.; Vincent, K. A.; Armstrong, F. A. Unusual reaction of [NiFe]-hydrogenases with cyanide. Journal of the American Chemical Society 2014, 136 , 10470-10477. (9) Ahn, S. D.; Fisher, A. C.; Buchard, A.; Bull, S. D.; Bond, A. M.; Marken, F. Hydrodynamic Rocking Disc Electrode Study of the TEMPO‐mediated Catalytic Oxidation of Primary Alcohols. Electroanalysis 2016, 28 , 2093-2103. (10) Elouarzaki, K.; Fisher, A. C.; Lee, J.-M. Molecular porphyrinic freestanding buckypaper electrodes from carbon nanotubes for glucose fuel cells. Journal of Materials Chemistry A 2017, 5, 8927-8932. (11) Vincent, K. A.; Parkin, A.; Lenz, O.; Albracht, S. P.; Fontecilla-Camps, J. C.; Cammack, R.; Friedrich, B.; Armstrong, F. A. Electrochemical definitions of O2 sensitivity and oxidative inactivation in hydrogenases. Journal of the American Chemical Society 2005, 127, 18179-18189. (12) Ceccaldi, P.; Marques, M. C.; Fourmond, V.; Pereira, I. C.; Léger, C. Oxidative inactivation of NiFeSe hydrogenase. Chemical Communications 2015, 51, 14223-14226. (13) Orain, C.; Saujet, L.; Gauquelin, C.; Soucaille, P.; MeynialSalles, I.; Baffert, C.; Fourmond, V.; Bottin, H.; Léger, C. Electrochemical measurements of the kinetics of inhibition of two FeFe hydrogenases by O2 demonstrate that the reaction is partly reversible. Journal of the American Chemical Society 2015, 137 (39), 12580-12587. (14) Megarity, C. F.; Esselborn, J.; Hexter, S. V.; Wittkamp, F.; Apfel, U.-P.; Happe, T.; Armstrong, F. A. Electrochemical Investigations of the Mechanism of Assembly of the Active-site HCluster of [FeFe]-hydrogenases. Journal of the American Chemical Society 2016, 138, 15227-15233. (15) Limoges, B. t.; Savéant, J.-M. Catalysis by immobilized redox enzymes. Diagnosis of inactivation and reactivation effects through odd cyclic voltammetric responses. Journal of Electroanalytical Chemistry 2004, 562, 43-52. (16) Savéant, J.; Su, K. Homogeneous redox catalysis of electrochemical reaction: Part VI. Zone diagram representation of the kinetic regimes. Journal of electroanalytical chemistry and interfacial electrochemistry 1984, 171, 341-349. (17) Artero, V.; Saveant, J.-M. Toward the rational benchmarking of homogeneous H 2-evolving catalysts. Energy & environmental science 2014, 7, 3808-3814. (18) Jones, A. K.; Lamle, S. E.; Pershad, H. R.; Vincent, K. A.; Albracht, S. P.; Armstrong, F. A. Enzyme electrokinetics: electrochemical studies of the anaerobic interconversions between active and inactive states of Allochromatium vinosum [NiFe]hydrogenase. Journal of the American Chemical Society 2003, 125 (28), 8505-8514. (19) Lukey, M. J.; Parkin, A.; Roessler, M. M.; Murphy, B. J.; Harmer, J.; Palmer, T.; Sargent, F.; Armstrong, F. A. How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. Journal of Biological Chemistry 2010, 285, 3928-3938. (20) Fourmond, V.; Infossi, P.; Giudici-Orticoni, M.-T. r. s.; Bertrand, P.; Léger, C. “Two-step” chronoamperometric method for studying the anaerobic inactivation of an oxygen tolerant NiFe hydrogenase. Journal of the American Chemical Society 2010, 132 (13), 4848-4857. (21) Elouarzaki, K.; Le Goff, A.; Holzinger, M.; Thery, J.; Cosnier, S. Electrocatalytic Oxidation of Glucose by Rhodium PorphyrinFunctionalized MWCNT Electrodes: Application to a Fully Molecular Catalyst-Based Glucose/O2 Fuel Cell. Journal of the American Chemical Society 2012, 134, 14078-14085, DOI: 10.1021/ja304589m. (22) De Lacey, A. L.; Fernández, V. M.; Rousset, M.; Cammack, R. Activation and Inactivation of Hydrogenase Function and the Catalytic Cycle: Spectroelectrochemical Studies. Chemical Reviews 2007, 107, 4304-4330, DOI: 10.1021/cr0501947.



(23) Mazurenko, I.; Monsalve, K.; Rouhana, J.; Parent, P.; Laffon, C.; Goff, A. L.; Szunerits, S.; Boukherroub, R.; Giudici-Orticoni, M.-T.; Mano, N.; Lojou, E. How the Intricate Interactions between Carbon Nanotubes and Two Bilirubin Oxidases Control Direct and Mediated O2 Reduction. ACS Applied Materials & Interfaces 2016, 8, 23074-23085, DOI: 10.1021/acsami.6b07355. (24) Plumeré, N.; Rüdiger, O.; Oughli, A. A.; Williams, R.; Vivekananthan, J.; Pöller, S.; Schuhmann, W.; Lubitz, W. A redox hydrogel protects hydrogenase from high-potential deactivation and oxygen damage. Nature Chemistry 2014, 6, 822, DOI: 10.1038/nchem.2022 https://www.nature.com/articles/nchem.2022#supplementaryinformation. (25) Rengaraj, S.; Haddad, R.; Lojou, E.; Duraffourg, N.; Holzinger, M.; Le Goff, A.; Forge, V. Interprotein Electron Transfer between FeS-Protein Nanowires and Oxygen-Tolerant NiFe Hydrogenase. Angewandte Chemie International Edition 2017, 56, 7774-7778, DOI: 10.1002/anie.201702042. (26) Volbeda, A.; Martin, L.; Liebgott, P.-P.; De Lacey, A. L.; Fontecilla-Camps, J. C. [NiFe]-hydrogenases revisited: nickel– carboxamido bond formation in a variant with accrued O2tolerance and a tentative re-interpretation of Ni-SI states. Metallomics 2015, 7, 710-718, DOI: 10.1039/C4MT00309H. (27) Luo, X.; Brugna, M.; Tron-Infossi, P.; Giudici-Orticoni, M. T.; Lojou, E. Immobilization of the hyperthermophilic hydrogenase from Aquifex aeolicus bacterium onto gold and carbon nanotube electrodes for efficient H 2 oxidation. JBIC Journal of Biological Inorganic Chemistry 2009, 14, 1275-1288. (28) Costentin, C.; Porter, T. R.; Savéant, J.-M. How Do Pseudocapacitors Store Energy? Theoretical Analysis and Experimental Illustration. ACS Applied Materials & Interfaces 2017, 9, 8649-8658. (29) Bard, A. J.; Faulkner, L. R. Fundamentals and applications, 2001; Vol. 2. (30) Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry 1979, 101, 19-28. (31) Stevenson, G. P.; Lee, C.-Y.; Kennedy, G. F.; Parkin, A.; Baker, R. E.; Gillow, K.; Armstrong, F. A.; Gavaghan, D. J.; Bond, A. M. Theoretical analysis of the two-electron transfer reaction and experimental studies with surface-confined cytochrome c

Page 12 of 14

peroxidase using large-amplitude Fourier transformed AC voltammetry. Langmuir 2012, 28, 9864-9877. (32) Crank, J.; Nicolson, P. In A practical method for numerical evaluation of solutions of partial differential equations of the heatconduction type, Mathematical Proceedings of the Cambridge Philosophical Society, Cambridge University Press: 1947; pp 5067. (33) Tse, Y. H.; Seymour, P.; Kobayashi, N.; Lam, H.; Leznoff, C. C.; Lever, A. Surface electrochemistry of chloro (phthalocyaninato) rhodium (III) species and oxygen reduction electrocatalysis. Formation of a dimeric species. Inorganic Chemistry 1991, 30, 4453-4459. (34) Thompson, C. M.; Shure, L. Image processing toolbox: User's guide, The MartWorks: 1995. (35) Mocak, J.; Feldberg, S. W. The Richtmyer modification of the fully implicit finite difference algorithm for simulations of electrochemical problems. Journal of Electroanalytical Chemistry 1994, 378, 31-37. (36) Crank, J.; Nicolson, P. A practical method for numerical evaluation of solutions of partial differential equations of the heatconduction type. Advances in Computational Mathematics 1996, 6 (1), 207-226. (37) Compton, R. G.; Banks, C. E. Understanding voltammetry, World Scientific: 2011. (38) Untereker, D.; Bruckenstein, S. Interpretation of isopotential points. Common intersection in families of current-potential curves. Analytical Chemistry 1972, 44 (6), 1009-1020. (39) Gaudiello, J. G.; Wright, T. C.; Jones, R. A.; Bard, A. J. Electrochemical reduction and reoxidation accompanied by reversible geometric isomerization. Electrochemistry of bis (dicarbonyl-. mu.-di-tert-butylphosphidorhodium). Isolation and xray crystal structure of bis (tetra-n-butylammonium) bis (dicarbonyl-. mu.-di-tert-butylphosphidorhodate). Journal of the American Chemical Society 1985, 107 (4), 888-897. (40) Armstrong, F. A.; Evans, R. M.; Hexter, S. V.; Murphy, B. J.; Roessler, M. M.; Wulff, P. Guiding principles of hydrogenase catalysis instigated and clarified by protein film electrochemistry. Accounts of chemical research 2016, 49 (5), 884-892.

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Hydrogenase-Like Electrocatalytic Activation and Inactivation

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