The oxidation-reduction and electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans

Journal Pre-proof The oxidation-reduction and Electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans

Palraj Kalimuthu, Mélanie Petitgenet, Dimitri Niks, Stephanie Dingwall, Jeffrey R. Harmer, Russ Hille, Paul V. Bernhardt PII:

S0005-2728(19)30167-7

DOI:

https://doi.org/10.1016/j.bbabio.2019.148118

Reference:

BBABIO 148118

To appear in:

BBA - Bioenergetics

Received date:

26 August 2019

Revised date:

19 October 2019

Accepted date:

4 November 2019

Please cite this article as: P. Kalimuthu, M. Petitgenet, D. Niks, et al., The oxidationreduction and Electrocatalytic properties of CO dehydrogenase from Oligotropha carboxidovorans, BBA Bioenergetics(2019), https://doi.org/10.1016/ j.bbabio.2019.148118

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© 2019 Published by Elsevier.

Journal Pre-proof

The Oxidation-Reduction and Electrocatalytic Properties of CO Dehydrogenase from Oligotropha carboxidovorans Palraj Kalimuthu,a Mélanie Petitgenet,a Dimitri Niks,b Stephanie Dingwall,b Jeffrey R. Harmer,c Russ Hilleb and Paul V. Bernhardta,* School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia

b

Department of Biochemistry, University of California, Riverside, CA 92521, USA

c

Centre for Advanced Imaging, University of Queensland, Brisbane 4072, Australia

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a

Abstract

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CO dehydrogenase (CODH) from the Gram-negative bacterium Oligotropha carboxidovorans is a complex metalloenzyme of the xanthine oxidase family of molybdenum-containing enzymes, bearing

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a unique bimolecular Mo-S-Cu active site in addition to two [2Fe-2S] clusters (FeSI and FeSII) and one equivalent of FAD. CODH catalyzes the oxidation of CO to CO2 with the concomitant introduction of

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reducing equivalents into the quinone pool, thus enabling the organism to utilize CO as sole source of both carbon and energy. Using a variety of EPR monitored redox titrations and

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spectroelectrochemistry, we report the redox potentials of CO dehydrogenase at pH 7.2 as MoVI/V, MoV/IV, FeSI , FeSII, FAD/FADH and FADH/FADH- vs NHE. These potentials are systematically higher than the corresponding potentials seen for other members of the xanthine oxidase family of Mo enzymes, and are in line with CODH utilising the higher potential quinone pool as an electron acceptor instead of pyridine nucleotides. CODH is also active when immobilised on a modified Au working electrode as demonstrated by cyclic voltammetry in the presence of CO.

Keywords: molybdenum, enzyme, redox potential, voltammetry

*

Corresponding author: E-mail address: [email protected] (P.V. Bernhardt)

Journal Pre-proof 1.

Introduction Oxidation of carbon monoxide (CO) to carbon dioxide (CO2) by aerobic carboxydotrophic

bacteria has been estimated to be on the scale of 200 megatonnes per annum [1, 2]. The enzyme responsible for this globally important reaction is a Mo-dependent CO dehydrogenase (CODH). CODH has been identified in several aerobic bacteria [3-6] of which the most intensively studied to date is the enzyme from Oligotropha carboxidovorans [7-15]. CODH, which was first isolated and characterised almost 40 years ago [14], is a 277 kDa (βγ)2 hexamer that belongs to the xanthine oxidase family of molybdenum-containing enzymes [16, 17]. However, it stands out from the

comprising a Mo-S-Cu moiety [7], which is unique to CODH.

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otherwise mononuclear (pterin-dependent) Mo enzymes as it bears a binuclear active site

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Like most other members of the xanthine oxidase family, CODH also possesses two [2Fe-2S] clusters and a flavin adenine dinucleotide (FAD) cofactor, which relay electrons from the Mo active

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site after CO oxidation to the quinone pool [18]. The spatial relationship between the four cofactors

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within each CODH protomer, as determined by X-ray crystallography [7], is shown in Figure 1.

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FeSI

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FeSII

FAD

Mo

Cu Figure 1. The redox-active cofactors within the CODH βγ protomer: from left to right, the Mo-S-Cu active site, FeSI, FeSII and FAD. Coordinates taken from its published [7] crystal structure (PDB 1N5W).

No proteins have been identified as physiological electron acceptors from O. carboxidovorans CODH, and NAD(P)+ or dioxygen cannot be reduced by CODH [14]. On the other

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Journal Pre-proof hand, a variety of quinones are effective oxidizing substrates, leading to the current hypothesis that CODH, which is associated with the inner plasma membrane, donates electrons to the quinone pool [18]. This suggests that the redox-active centers of the enzyme might have higher redox potentials than is typical for members of the xanthine oxidase family, which normally reduce pyridine nucleotides or dioxygen. In this work we report the electrochemical characterisation of all redox-active centers in CODH

using

a

combination

of

EPR-monitored

redox

potentiometry

and

optical

spectroelectrochemistry. In addition, we demonstrate that CODH can be electrochemically activated

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for CO oxidation through cyclic voltammetry.

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Experimental Section

2.1 Materials 2.1.1 Protein Purification CODH was obtained from O. carboxidovorans cells (ATCC 49405) and purified as described previously [9]. As isolated, CO oxidation activity is typically poor due to low incorporation of the catalytically essential Cu and bridging sulfido ligand of the binuclear active site. Reconstitution was achieved using the protocol of Resch et al. [8] comprising incubation with Na2S under reducing conditions followed by CuI incorporation via its thiourea complex. The final Cu loading yielded

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enzyme that was approximately 50% functional. 2.1.2 Reagents

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All synthetic reagents, solvents, buffers and gases were obtained commercially and used

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without further purification.

2.2 Methods

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2.2.1 EPR Potentiometry

CODH (1.2 mL, ~90 μM) was mixed with glycerol (100 μL) and 10 μL of 4 mM solutions of the

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mediators methylene blue, 2,5-dihydroxybenzene, 2-hydroxynaphthoquinone, anthraquinone-2sulfonate, indigo-5,5’,7-trisulfonate, benzyl viologen and methyl viologen. The solutions were

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titrated with ca. 10 mM solutions of sodium dithionite (reductant) or sodium persulfate (oxidant) within a Belle Technology anaerobic glovebox (O2 < 20 ppm) until a stable oxidation-reduction

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potential (ORP) was achieved. The equilibrium ORP was measured with a combination Pt wire and Ag/AgCl electrode calibrated with quinhydrone (Em,7 = +285 mV vs NHE). All potentials are given vs NHE. Upon equilibration, a 50 μL aliquot was withdrawn and transferred to an EPR tube, which was sealed with a rubber septum and frozen in liquid nitrogen immediately after removal from the glovebox. X-band (ca. 9 GHz) continuous-wave (CW) EPR spectra were acquired at 130 or 20 K using a Bruker Elexsys E540 or E580 spectrometer equipped with an ElexSys Super High Sensitivity Probehead (E540) or Bruker MD5 flexLine resonator (E580) and either liquid N2 (E540 with Eurotherm temperature control) or liquid He (E580 with a cryogen-free system from Cryogenics) cooling.. The magnetic field was calibrated with 2,2-diphenyl-1-picrylhydrazyl (g = 2.0036). The potential dependent EPR intensities I(E) were fit to either equation 1a for a single electron process (FeSI and FeSII, redox potential E1) or equation 1b for a two electron reduction (potentials E1 and E2) where the single electron reduced form is the only EPR active species (MoV); Imax is the maximum signal intensity of the EPR active species and E is the measured ORP (mV).

4

Journal Pre-proof 𝐼𝑚𝑎𝑥

𝐼(𝐸) =

1 + 10 𝐼(𝐸) = 1+

(1𝑎)

𝐸−𝐸1 59

𝐼𝑚𝑎𝑥 𝐸−𝐸1 𝐸2−𝐸 10 59 + 10 59

(1𝑏)

2.2.2 Optical Spectroelectrochemistry Experiments were conducted with a Pine Instruments quartz spectroelectrochemical cell (1.7 mm optical path length) using a Pt ‘honeycomb’ working electrode, a Pt auxiliary electrode and a Ag/AgCl reference electrode calibrated with quinhydrone as mentioned above. The solutions were approximately 33 μM in CODH (66 μM in FAD) in HEPES buffer (50 mM, pH 7.2). To avoid

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interference from highly colored organic meditators each experiment contained 20 μM of the

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complexes [Fe(NOTA)], [Fe(tacn)2]3+, [Co((NMe3)2sar)]5+, [Co(CLMEN4S2sar)]3+, [Co(AMMEN5Ssar)]3+, and [Co(sep)]3+ [19] (see supporting information Figure S1 for structures). These complexes span the

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redox potential range +200 > Em,7 > -300 mV vs NHE and have small molar extinction coefficients (< 300 M-1cm-1) so they make no contribution to the visible spectra at micromolar concentrations.

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Spectra were acquired within an anaerobic glovebox with an Ocean Optics USB2000 fibre optic

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spectrophotometer and a DT-MINI-2-GS miniature deuterium/tungsten/halogen UV-Vis-NIR light source. Potentials were set with a BAS100B/W potentiostat operating in chronocoulometry mode and UV-Vis spectra were taken when equilibrium was established and all absorbance changes ceased

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(typically within 10 min). Spectra were taken at 50 mV intervals and reversibility was established by stepping the potential firstly in negative and then in positive directions. No significant hysteresis was

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found. Data were modelled with Reactlab Redox [20] using a two electron transfer model (similar to equation 1b) which produced the two single electron redox potentials as well as the spectra of the fully oxidised (FAD, quinone), single electron reduced (FADH• semiquinone) and fully reduced (FADHhydroquinone) forms.

2.2.3 Cyclic Voltammetry All electrochemical experiments were carried out at 25°C with a BAS100B potentiostat and BAS RDE-2 rotating disk cell stand either in stationary or rotating mode as described. The auxiliary electrode was a Pt wire and a Ag/AgCl reference electrode was used. The electrolyte was 50 mM HEPES buffer at pH 7.2 or, for pH-dependent experiments, a mixture of buffers (25 mM Bis-Tris buffer pH 5.8–7.2, 25 mM Tris buffer pH 7.0–9.0 and 25 mM CHES buffer pH 8.6–10.0) titrated with dilute HCl or NaOH to the desired pH.

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Journal Pre-proof 2.2.4 Enzyme Immobilisation A ca. 3 mm Au disk electrode (surface area 0.055 cm2) was cleaned as described [21] then soaked in EtOH for 1 h. The electrode was then transferred to a solution of dithiobis(succinimidyl propionate) (DSP) (8 mg in 1 mL DMSO) and allowed to react for 2 h. The electrode was washed thoroughly with acetone and air dried. A mixture of CODH (3 μL, 20 μM) and chitosan (3 μL, 0.1% in 1% AcOH) was carefully pipetted onto the inverted Au electrode then the droplet was allowed to dry to a film at room temperature. At the completion of the experiment, reductive desorption of the thiol modified Au electrode found the total coverage of Au-S bonds to be in the range 3-6 × 10-11 mol. To prolong the activity of the Au-DSP-CODH electrode in pH-dependent experiments a 1 cm2

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dialysis membrane (MW cutoff 3500 Da) pre-soaked in water was carefully placed over the electrode

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and fastened with a rubber O-ring.

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Results and Discussion

3.1 Redox Potential Measurements 3.1.1 EPR Redox Titrations The cofactors in CODH have been well characterised by EPR spectroscopy [8, 9, 22] but their redox potentials have not been reported. In order to avoid spectral overlap during redox titrations, EPR spectra (at a given potential) were recorded at different temperatures and microwave powers in order to isolate the signals of the MoV, FADH•, FeSI and FeSII centers. The EPR signals of MoV and FADH• centers are readily observed at liquid N2 temperatures, while the [2Fe-2S] clusters, with their shorter spin relaxation times, required much lower temperatures (< 40 K) to be observed. This

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meant that the temperature- and potential-dependent EPR spectra were acquired over a period of

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days and even weeks in some cases. Re-CW measurement of the 130 K EPR spectra up to eight weeks after the first experiment showed no differences, indicating that the samples stored in liquid

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nitrogen were stable on the long term and were not susceptible to reoxidation by O2. As CODH is a (βγ)2 hexamer and the redox-centers of each protomer are well-separated from one another [7], it

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was assumed that these pairs of related cofactors were reduced and oxidised independently and at

oxidase family [23]. 3.1.1.1 The binuclear Mo-S-Cu center

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the same potentials. This is a well-established convention in studies of enzymes of the xanthine

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The Mo-S-Cu site is only EPR-active in its MoV oxidation state. Although no potentialdependent EPR spectra have previously been reported for CODH, the CuI ion is believed to be redox-

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inert, which would be consistent with its enforced approximately linear CuIS2 coordination geometry. Although the redox-active orbital of the binuclear cluster is formally the singly occupied Mo 4dxy orbital, studies on structural models of the active site have shown that there is substantial unpaired electron density on the Cu [24], which is thought to be critical to the reactivity of the center [25]. The MoV-S-Cu signal features strong and anisotropic hyperfine coupling of the unpaired electron with the Cu nucleus (I = 3/2, A1,2,3 117, 164, 132 MHz; [9]) to produce a set of four peaks each split into their x, y and z components by the rhombic symmetry of the active site. An example is shown in Figure 2A which matches the spectrum published for the dithionite-reduced enzyme [9]. However, various inactive forms of the Mo active site may also be seen if the labile Cu or sulfido ligand are absent [8, 26]. Indeed, a rhombic MoV EPR signal with g values ~2.0, 1.96 and 1.93 was observed at high potentials (above -100 mV vs NHE, Supporting Information Figure S2) and its MoV signal is maximal around -25 mV. The lack of hyperfine coupling indicates that the Cu is absent from this form of CODH and this signal has been attributed to the (MCD)MoVO3 form which has lost both the sulfido ligand and the Cu ion [8].

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Journal Pre-proof Of principal interest here was the potential-dependent EPR spectrum of the functional MoV-S-CuI center. This MoV signal emerged around -100 mV and remained constant over a wide range in potential before declining below -300 mV and completely disappearing by -400 mV, where the EPR silent MoIV-S-CuI form presumably dominated (Figure 2B). The intensities were fit to equation 1b to yield redox potentials of -165 mV and -358 mV vs NHE for the MoVI/V MoV/IV couples, respectively (pH 7.2).

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0.5

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Mo(V) intensity

1.0

310

320

330

340

350

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0.0

360

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B (mT)

-400

-300

-200

-100

0

E (mV vs NHE)

3.1.1.2 FeSI

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Figure 2. (A) X-band (9.377 GHz) CW EPR spectrum of CODH at 130 K poised at -259 mV vs NHE showing the MoV-S-Cu signal (B) redox titration results monitoring the MoV-S-Cu signal at 355 mT of CODH (pH 7.2). The solid line is a fit to equation (1b) with values -165(±13) and -358(±12) mV vs NHE.

It is known that FeSI (the cluster closer to the active site) yields an intense and well resolved rhombic signal (g values 2.023, 1.945 and 1.900) below 40 K upon reduction to the mixed valent (FeIII/FeII) state [22, 26]. As shown in Figure 3A, the 20 K spectrum of the enzyme poised at -166 mV vs NHE and 46 dB microwave power, (conditions under which all MoV signals are saturated) is dominated by the signal of FeSI, although the FADH• signal persists to some extent. Representative spectra appear in the Supporting Information Figure S3. The EPR signal of the FeSII cluster is absent at this potential (see below). The dependence of the signal on oxidation-reduction potential is shown in Figure 3B, yielding a redox potential of -116 ± 8 mV; the FeSI signal develops at a potential 100-200 mV higher than seen in other members of the xanthine oxidase family (Table 1).

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B

A FADH.

FeSI intensity (g = 1.945)

g1 = 2.023

g2 = 1.945

g3 = 1.900

330

340

3

350

360

370

2

1

0 -400 -300 -200 -100

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B (mT)

0

100 200 300

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E (mV vs NHE)

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Figure 3. (A) X-band (9.671 GHz) CW EPR spectrum (20 K) of the FeSI cofactor poised at -166 mV vs NHE (B) redox titration of the FeSI signal. The solid line is a fit to equation (2) with a value of Em,7.2 -116(±8) mV vs NHE.

Table 1. Redox potentials (mV vs NHE) of selected enzymes from the xanthine oxidase family. pH

FAD/ FADH• -350

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source

FADH• /FADH-350

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xanthine P. putida 8.0 dehydrogenase xanthine R. capsulatus 8.0 -410 -484 dehydrogenase xanthine chicken liver 7.8 -345 -377 dehydrogenase xanthine turkey liver 8.2 -359 -362 dehydrogenase xanthine oxidase cow’s milk 7.7 -332 -234 aldehyde rabbit liver 7.4 -133a oxidase CO O. carboxi7.2 -18 -133 dehydrogenase dovorans a averaged midpoint potential; n.d. not determined

FeSII

MoVI/V

MoV/IV

-335

n.d.

-360

-300

[27]

-256

n.d.

-421

-499

[28]

-280

-275

-357

-337

[29]

-295

-292

-350

-362

[30]

-310 -207

-255 -310

-373 -294

-377 -468

[31] [32]

-116

-268

-165

-357

this work

FeSI

Ref.

3.1.1.3 FeSII The EPR signal from this cluster in CODH is unusual in showing a greater anisotropy and much faster spin relaxation rate than FeSI [22, 26]. This has also been reported for other members of the xanthine oxidase family including mammalian xanthine oxidase [33] and bacterial xanthine dehydrogenase [34]. Figure 4 illustrates the spectrum of a sample where both the FeSI and FeSII centers are reduced and EPR active. At lower power 0.00498 mW (46 dB of 200 mW) only the FeSI

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Journal Pre-proof signal is seen (Figure 4A, upper trace) but upon an increasing the power to 4.98 mW (16 dB of 200 mW) (Figure 4A, lower trace) the reduced FeSII center emerges as the FeSI signal begins to saturate, although two of its three g values overlap with those for the FeSI spectrum. Still, the FeSII cluster exhibits a very high g1 value (2.156) which is well clear of the more intense FeSI g1, thus enabling its accurate quantification as a function of potential. Figure S4 (Supporting Information) illustrates the spectral changes at high power with potential. Notably, the redox potential for FeSII is some -150 mV more negative than that of the FeSI cluster.

g1 = 2.023

g2 = 1.945 x5 g3 = 1.900

g1 = 2.156

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5

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g3 = 1.901

0

-400

-300

-200

-100

0

E (mV vs NHE)

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310 320 330 340 350 360 370 380 B (mT)

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g2 = 2.009

20

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B

BV.+

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46dB 16dB

FeSII intensity (g = 2.156)

A

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Figure 4. (A) X-band (9.655 GHz) CW EPR spectrum of the FeSI and FeSII centers (20 K) poised at -248 mV vs NHE as a function of microwave power showing the emergence of the FeSII signals at high power (4.98 mW, 16 dB of 200 mW), the benzyl viologen (BV) radical signal is saturated at this higher power (B) redox titration following the FeSII signal at 320 mT. The solid line is a fit to equation (2) with a value of Em,7.2 -268(±5) mV vs NHE.

3.1.2 Spectroelectrochemistry 3.1.2.1 FAD Cofactor

Although the FADH• semiquinone is detectable by EPR (see Figure 3A), the potential window over which it is seen is quite narrow i.e. the FAD/FADH• and FADH•/FADH- half-potentials are relatively close together. The EPR spectrum of the FADH• semiquinone of CODH has been observed previously at pH 6 [35] where the two couples are presumably better separated; the higher potential FAD/FADH• couple being shifted positively due to an associated protonation equilibrium. For consistency, all of the measurements in this work were at pH 7.2, so the FAD EPR signal was only seen fleetingly during the course of the potentiometric titration. Moreover, overlap of the FADH•

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Journal Pre-proof signal (g ~2.0) with signals from both the active and inactive forms of the molybdenum center (See Figure S2) complicated determination of the two flavin half-potentials by EPR alone. As a practical alternative, we employed optical spectroelectrochemistry to take advantage of the strong absorption of the FAD (454 nm, ~35,000 M-1 cm-1) in its oxidised (FAD) state.

0.30

FADox FADsq FADred

Abs.

0.20

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50000

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 (M-1 cm-1)

0.25

100000 abs@508 nm abs@610 nm

500

550

600

650

-p

0.08

-0.40 -0.30 -0.20 -0.10

0.00

0

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0.07 0.10

E (V vs NHE)

400 450 500 550 600 650 700 750 800 wavelength (nm)

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Figure 5. (A) Optical spectroelectrochemistry results for CODH (33 μM, pH 7.2) showing (A) the changes in absorbance at 610 nm (squares) and 508 nm (circles) as a function of potential; solid lines are calculated with redox potentials of -18 mV (FAD/FADH•) and -133 mV vs NHE (FADH•/FADH-) and (B) the calculated spectra (Reactlab Redox) in the oxidised, one-electron reduced and two-electron reduced forms.

The potential-dependent spectral changes at 508 and 610 nm are shown in Figure 5A. All of the potential dependent spectra appear in the Supporting Information (Figure S5). The features to note are an approximately monotonic decrease in the 508 nm absorbance as the potential is lowered, and rise-fall behaviour in the absorbance at 610 nm, reflecting the transient accumulation of the long-wavelength absorbing FADH•. This is better appreciated in the calculated spectra of the FAD, FADH• and FADH- forms (Figure 5B) which show the expected decrease in the 454 nm absorption peak with reduction but an increase around 600 nm indicative of the neutral FADH• intermediate; the fully reduced FADH- spectrum being essentially featureless. Similar spectral features have been reported for the quantitative dithionite reduction of CODH [9] but without redox potential measurement. It should be noted that both (oxidised) [2Fe-2S] clusters absorb around 500 nm so some of the spectral changes at 508 nm could be attributable to [2Fe-2S] (FeSI) reduction. However, at 610 nm the Fe-S clusters do not absorb significantly and their reduction could not in any

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Journal Pre-proof case account for the observed transient increase in absorbance. Therefore, the changes at this wavelength are principally due to the formation and decay of the FADH• radical.

3.2 Cyclic Voltammetry 3.2.1 Mediator Selection The spectroelectrochemical results show that the level of reduction of the several redoxactive centers of CODH may be controlled potentiostatically by mediators of a suitable redox potential. Ideally, if poised at its fully oxidised state the enzyme should sustain CO oxidation under electrochemical conditions. On the basis of the redox potentials determined above, a range of suitable electroactive mediators were chosen for electrocatalytic CO turnover experiments (Figure

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6). Given the solvent-exposed position of the FAD cofactor, it is the obvious site of electron egress

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from CODH, so the FAD redox potentials set the lower potential bounds of appropriate artificial electron acceptors. The mediators shown in Figure 6 have suitable redox potentials to act as

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the natural electron acceptor of the enzyme.

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oxidants of CODH and also possess structures that resemble the aromatic quinones thought to be

Figure 6. Organic redox mediators examined in this work.

3.2.2 Enzyme Immobilisation It was impractical to study CODH solutions at sufficiently high concentrations to observe a mediated electrochemical reaction directly. On the other hand, a monolayer of enzyme can give rise to measurable voltammetry currents particularly under turnover conditions when amplified by substrate turnover. However, this method relies on the formation of an active and stable enzyme film on the electrode surface. We addressed the issue of film stability by covalently attaching a composite of the poly-glucosamine chitosan and CODH to the Au electrode surface. The modification

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Journal Pre-proof of Au electrode surfaces with a self-assembling monolayer of organic thiols, to avoid protein denaturation at the bare Au surface, is a well-established area of research [36] and various protocols have been reported [21, 37, 38]. Bifunctional thiols have also been employed to provide direct covalent attachment of proteins via their lysine residues [39, 40]. The methodology herein utilised the bifunctional linker dithiobis(succinimidyl propionate) (DSP) [40], which forms amide linkages with primary amino groups non-specifically; either from surface lysine residues of CODH or the polymeric co-adsorbate chitosan, while the thiol groups bind to the electrode surface via strong Au-S bonds. Any succinimide esters that do not condense with the available primary amines are hydrolysed to leave propionate chains ionised at pH 7.2. Although

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the actual amount of CODH immobilised on the electrode could not be quantified, due to the

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absence of non-turnover responses, the coverage of 3-mercaptopropionate chains on the Au surface could be determined by quantitative reductive desorption of the layer. The coverage of thiols from

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multiple experiments was around 3 × 10-11 mol on the 0.055 cm2 electrode (5.5 × 10-10 mol cm-1), which is sub-monolayer coverage by comparison with long chain alkanethiol-modified Au electrode

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studies (~ 8 × 10-10 mol cm-2) [41].

3.2.3.1 CO Dependence

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3.2.3 Methylene Blue-Mediated Voltammetry

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Given the well-established activity of CODH with methylene blue (MB) as an electron

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acceptor [14], the main focus of our electrochemistry was with MB as the redox-active mediator.

1500 M CO

2 A

750 M CO

375 M CO 188 M CO 94 M CO 0 M CO

-150 -100 -50

0

50 100 150 200 250

E (mV vs NHE)

Figure 7. Cyclic voltammograms of the stationary Au-DSP-CODH electrode in the presence of methylene blue (50 μM) at various CO concentrations. Scan rate 10 mV s-1, 50 mM HEPES buffer pH 7.2.

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In Figure 7 (dark red-brown curve) the CV of MB (50 μM) at the Au-DSP-CODH electrode in the absence of CO is shown. The typical reversible two-electron response of MB appears at a potential of +10 mV vs NHE (pH 7.2) which is consistent with its known electrochemical properties [42]. Saturating the solution with CO ([CO] = 1500 μM) results in an approximately sigmoidal response with an amplified anodic current (Figure 7, light blue curve). The characteristic amplification of anodic current (MB oxidation) upon addition of CO is due to continual reduction of MB by CODH following reduction of the enzyme by CO while the reducing equivalents thus

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generated are relayed by MB into the electrical circuit and measured as current. Successive two-fold dilutions of this solution with argon-saturated HEPES buffer containing

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50 μM MB led to a decrease in the observed current and a change to a peak-shaped waveform. It is notable that in the presence of CO ([CO] > 94 μM) the initial rising portion of the anodic waves are

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identical. That is, the CO turnover rate is enzyme-limited over this potential range and independent

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of CO concentration because the enzyme is saturated with substrate (KM,CO = 11 μM) [9]. The decrease in current beyond the peak is due to mass transport limitations of CO reaching the

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electrode surface. Although rotating disk voltammetry could in principle obviate this problem, electrode rotation leads to a faster rate of protein film degradation than with a stationary electrode

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(Supporting Information Figures S6 and S7), and better ways to stabilise the film are needed to prolong the activity of the electrode.

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3.2.3.2 Enzyme Film Stability

Experiments with Au-DSP-CODH electrodes prepared without chitosan led to almost immediate loss of electro-catalytic activity (Supporting Information Figure S8). Furthermore, on standing, the initially blue 100 μM MB solution saturated with CO turned colourless (Supporting Information Figure S9), which shows that CODH desorbs and mixes in the electrochemical solution where it quantitatively reduces MB through CO oxidation. These observations suggest that chitosan is the key component that reacts preferentially with the succinimide esters of the Au-DSP electrode, while CODH is only noncovalently entrapped within the polymer. However, as will be shown, the time-dependent loss of Au-DSP-CODH electrode activity is due to a combination of CODH leaching from the enzyme-chitosan composite and specific loss of enzyme activity probably through dissociation of the catalytically essential Cu ion. 3.2.3.3 pH Dependence The CODH-chitosan composite was particularly unstable at mildly alkaline solutions. The primary amino groups of chitosan have average pKa values in the range 6.5-7.0 [43, 44], so the 14

Journal Pre-proof positive charges of the ammonium groups (which have not reacted with DSP) seem to be an important factor in stabilising the Au-DSP-CODH electrode. To prevent irreversible loss of CODH from the electrode surface to the bulk solution, the Au-DSP-CODH electrode was covered with a dialysis membrane (molecular weight cutoff 3,500 Da) for pH dependent experiments. Any decrease in activity under these conditions should be due specifically to enzyme deactivation and not enzyme film degradation. The disadvantage of using a membrane electrode is that diffusion of MB across the membrane is slow, making accurate determination of its concentration under the membrane problematic. This is illustrated in the Supporting Information Figure S10 where the response from

of

MB develops with successive cycles until the concentration under the membrane matches that of

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the bulk. Therefore, this equilibrium needs to be established before the pH-dependent experiments can begin. Due to its smaller size and greater mobility, CO diffuses more rapidly across the

pH 6.9 pH 7.4 pH 7.9 pH 8.3 pH 8.5 pH 9.0

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-100

-50

0

50

100

150

200

E (mV vs NHE)

catalytic current (relative to pH 7)

lP

re

1 A

na

A

-p

membrane and saturating concentrations of CO were always present.

1.2

B 1.0 0.8 0.6 0.4 0.2 0.0 5.5

6.0

6.5

7.0

7.5

8.0

8.5

pH

Figure 8. (A) Cyclic voltammograms of the Au-DSP-CODH electrode in the presence of methylene blue (100 μM) and CO (1.5 mM) at different pH values. Scan rate 10 mV s-1 and (B) catalytic currents from the Au-DSP-CODH electrode (covered with a membrane) normalised relative to pH 7.0 and corrected for loss of activity with time by re-measurement of activity periodically at pH 7.0.

The CVs in Figure 8A illustrate the Au-DSP-CODH membrane electrode activity across the range 6.9 < pH < 9.0. As expected, the wave shifts to higher potential as the pH is lowered due to the 2-electron, 1 proton transfer redox reaction of MB (Figure 8A). However, there is little apparent variation in the catalytic current. If the data are corrected for time-dependent degradation of the film by periodically remeasuring a standard solution (at pH 7.0) over the course of the experiment

15

Journal Pre-proof (Figure 8B), the catalytic currents may be normalised relative to the standard. Remarkably almost no pH dependence is seen across this range. The specific activity of CODH has been reported as a function of pH and similarly exhibits only a modest pH dependence across the range 5 < pH < 10 with activity never less than 60% of its optimum [9]. This is in contrast to the pH dependence of other enzymes from the xanthine oxidase family which typically show distinct and quite narrow bell shaped activity profiles [45, 46]. A Glu residue near the active site of CODH (E670) is universally conserved among members of the xanthine oxidase family and is generally thought to act as a base/nucleophile during catalysis; a recombinantly expressed CODH variant lacking this Glu residue (E670Q) was similarly inactive [13]. However, the lack of any comparable decrease in activity in

of

CODH in mildly acidic solution suggests that this Glu residue may play more than one role in catalysis. In other members of the xanthine oxidase family, the conserved Glu serves to deprotonate

ro

the catalytically labile equatorial Mo-OH that undertakes nucleophilic attack on substrate to initiate catalysis. In CODH, by virtue of the Mo-S-Cu linkage that replaces the Mo=S seen in other family

-p

members, the equatorial position is deprotonated as a second Mo=O group that need not (indeed

re

cannot) be deprotonated. Upon reduction of the binuclear center in the course of the reaction with CO, however, this equatorial position does become protonated to Mo-OH, and must be

lP

deprotonated in the course of reoxidation of the center for a subsequent round of catalysis to return to the Mo=O seen in the oxidized center. The Glu is well-positioned to facilitate this deprotonation,

na

which is likely to be essential for efficient electron egress from the binuclear center since, unlike most other members of the xanthine oxidase family, there is no large solvent access channel to the

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active site that would allow effective spontaneous deprotonation. 3.2.4 Alternative Mediators

Apart from MB, the remaining mediators in Figure 6 were also able to act as electron acceptors from CODH under electrochemical conditions. In the absence of CO, the electrochemistry of the mediators in Figure 9 (including MB for comparison) is well-behaved, and pairs of symmetrical oxidation-reduction peaks are found at pH 7.2 (Figure 9A-D, yellow curves). The redox responses of MB, N-methylphenazinium and 2,6-dichlorophenolindophenol (DCPIP) are all two-electron processes which are coupled to facile proton transfer reactions, while TMPD (N,N,N’,N’-tetramethyl-pphenylenediamine) undergoes a single electron oxidation to its radical cation. These responses are all amplified in the presence of a saturating concentration of CO, which is again indicative of mediated electrochemical catalysis. The successively higher catalytic potentials follow the redox potentials of the mediators.

16

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A

B

500 nA

500 nA

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0

-100 -50

50 100 150 200 250 300

0

50 100 150 200 250 300 E (mV vs NHE)

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E (mV vs NHE)

D

C

500 nA

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re

-p

ro

200 nA

E (mV vs NHE)

100 150 200 250 300 350 400 450 500

na

50 100 150 200 250 300 350 400 450

E (mV vs NHE)

4.

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Figure 9. Cyclic voltammograms of 50 μM solutions of (A) methylene blue, (B) N-methylphenazinium, (C) 2,6-dichlorophenolindophenol and (D) N,N,N’,N’-tetramethyl-p-phenylenediamine at a stationary Au-DSP-CODH electrode in the absence (light brown curves) and presence of CO (1.5 mM, dark blue curves). Scan rate 5 mV s-1, 50 mM HEPES buffer pH 7.2.

Conclusions

The redox potentials of O. carboxidovorans CODH reported here are systematically higher than those seen with other members of the xanthine oxidase family (Table 1). This feature is in line with the enzyme utilising high-potential quinone electron acceptors (ubiquinone, Em7 +66 mV vs NHE [47]) rather than pyridine nucleotides (NAD+, Em7 -309 mV vs NHE [48]) as seen with other members of the xanthine oxidase family. In particular, the FAD cofactor of CODH has the highest midpoint potential seen for a member of the xanthine oxidase family, some 275 mV more positive than that seen with the NAD+-dependent xanthine dehydrogenases from a variety of sources (Table 1). Similarly, the binuclear Mo/Cu center has a significantly higher midpoint potential than the mononuclear centers of other family members, principally due to a highly elevated half-potential for

17

Journal Pre-proof the MoVI/V couple. The MoV oxidation state is thus greatly stabilized thermodynamically, accounting for the ease with which the EPR-active state is generated experimentally. This stabilization is presumably due to the extensive delocalization of the redox-active Mo 4dxy orbital onto the copper of the binuclear center [24]. With regard to the iron-sulfur clusters, it is unusual for FeSI, which is proximal to the molybdenum center, to have a higher redox potential than FeSII; only the aldehyde oxidoreductase from D. gigas is similarly arranged [32]. However, it is the exception rather than the rule that the redox-active centers in multicenter proteins are laid out in systematically increasing or decreasing reduction potential. Most often, there is an unusually high- or low-potential center along the pathway, and it appears to not matter where specifically along the pathway the outlier is

of

positioned. This being the case, there is no reason a priori to expect one or another of the ironsulfur clusters to have the higher or lower potential, equilibration between them, and with the

ro

Mo/Cu and FAD, is simply fast enough to sustain catalysis.

-p

We find that upon immobilization on an Au electrode, CODH is an effective catalyst of CO oxidation in combination with electron transfer mediators. Catalysis is dependent on CO

re

concentration and saturates as anticipated at high CO concentrations. Under the conditions employed here (stationary electrode) the CVs show peak-shaped waveforms which indicates that

lP

mass transport of CO to the electrode is rate-limiting at all but saturated concentrations of CO. Typically, activity is sustained over a period of around 1 h at room temperature, with activity loss

na

due to a combination of film degradation and enzyme deactivation; the former process can be slowed by using a membrane electrode. In addition to methylene blue, a range of other organic

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mediators with higher redox potentials are also effective electron acceptors.

Acknowledgements

Financial support from the Australian Research Council to PVB (DP150103345) is gratefully acknowledged. This work was also supported by a grant from the US Department of Energy (DESC0010666) to RH.

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Journal Pre-proof Highlights

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 

characterisation of the redox active centres in CO dehydrogenase using potential dependent EPR and optical spectroscopy electro-catalytic CO oxidation mediated by methylene blue and other organic mediators pH and CO concentration dependence of CO dehydrogenase activity examines

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

21