Reduction of Carbon Dioxide by a Molybdenum-Containing Formate Dehydrogenase: A Kinetic and Mechanistic Study

Carbon dioxide accumulation is a major concern for the ecosystems, but its abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels. However, the thermodynamic and kinetic stability of the carbon dioxide molecule makes its activation a challenging task. Studying the chemistry used by nature to functionalize carbon dioxide should be helpful for the development of new efficient (bio)­catalysts for atmospheric carbon dioxide utilization. In this work, the ability of <i>Desulfovibrio desulfuricans</i> formate dehydrogenase (Dd FDH) to reduce carbon dioxide was kinetically and mechanistically characterized. The Dd FDH is suggested to be purified in an inactive form that has to be activated through a reduction-dependent mechanism. A kinetic model of a hysteretic enzyme is proposed to interpret and predict the progress curves of the Dd FDH-catalyzed reactions (initial lag phase and subsequent faster phase). Once activated, Dd FDH is able to efficiently catalyze, not only the formate oxidation (<i>k</i>_cat of 543 s^–1, <i>K</i>_m of 57.1 μM), but also the carbon dioxide reduction (<i>k</i>_cat of 46.6 s^–1, <i>K</i>_m of 15.7 μM), in an overall reaction that is thermodynamically and kinetically reversible. Noteworthy, both Dd FDH-catalyzed formate oxidation and carbon dioxide reduction are completely inactivated by cyanide. Current FDH reaction mechanistic proposals are discussed and a different mechanism is here suggested: formate oxidation and carbon dioxide reduction are proposed to proceed through hydride transfer and the sulfo group of the oxidized and reduced molybdenum center, Mo⁶⁺S and Mo⁴⁺-SH, are suggested to be the direct hydride acceptor and donor, respectively

Protein-Assisted Formation of Molybdenum Heterometallic Clusters: Evidence for the Formation of S₂MoS₂–M–S₂MoS₂ Clusters with M = Fe, Co, Ni, Cu, or Cd within the Orange Protein

The Orange Protein (ORP) is a small bacterial protein, of unknown function, that harbors a unique molybdenum/copper (Mo/Cu) heterometallic cluster, [S₂Mo^VIS₂Cu^IS₂Mo^VIS₂]^3–, noncovalently bound. The apo-ORP is able to promote the formation and stabilization of this cluster, using Cu^II- and Mo^VIS₄^2– salts as starting metallic reagents, to yield a Mo/Cu-ORP that is virtually identical to the native ORP. In this work, we explored the ORP capability of promoting protein-assisted synthesis to prepare novel protein derivatives harboring molybdenum heterometallic clusters containing iron, cobalt, nickel, or cadmium in place of the “central” copper (Mo/Fe-ORP, Mo/Co-ORP, Mo/Ni-ORP, or Mo/Cd-ORP). For that, the previously described protein-assisted synthesis protocol was extended to other metals and the Mo/M-ORP derivatives (M = Cu, Fe, Co, Ni, or Cd) were spectroscopically (UV–visible and electron paramagnetic resonance (EPR)) characterized. The Mo/Cu-ORP and Mo/Cd-ORP derivatives are stable under oxic conditions, while the Mo/Fe-ORP, Mo/Co-ORP, and Mo/Ni-ORP derivatives are dioxygen-sensitive and stable only under anoxic conditions. The metal and protein quantification shows the formation of 2Mo:1M:1ORP derivatives, and the visible spectra suggest that the expected {S₂MoS₂MS₂MoS₂} complexes are formed. The Mo/Cu-ORP, Mo/Co-ORP, and Mo/Cd-ORP are EPR-silent. The Mo/Fe-ORP derivative shows an EPR <i>S</i> = ³/₂ signal (<i>E</i>/<i>D</i> ≈ 0.27, <i>g</i> ≈ 5.3, 2.5, and 1.7 for the lower <i>M</i>= ±¹/₂ doublet, and <i>g</i> ≈ 5.7 and 1.7 (1.3 predicted) for the upper <i>M</i> = ±³/₂ doublet), consistent with the presence of either one <i>S</i> = ⁵/₂ Fe^III antiferromagnetically coupled to two <i>S</i> = ¹/₂ Mo^V or one <i>S</i> = ³/₂ Fe^I and two <i>S</i> = 0 Mo^VI ions, in both cases in a tetrahedral geometry. The Mo/Ni-ORP shows an EPR axial <i>S</i> = ¹/₂ signal consistent with either one <i>S</i> = ¹/₂ Ni^I and two <i>S</i> = 0 Mo^VI or one <i>S</i> = ¹/₂ Ni^III antiferromagnetically coupled to two <i>S</i> = ¹/₂ Mo^V ions, in both cases in a square-planar geometry. The Mo/Cu-ORP and Mo/Cd-ORP are described as {Mo^VI–Cu^I–Mo^VI} and {Mo^VI–Cd^II–Mo^VI}, respectively, while the other derivatives are suggested to exist in at least two possible electronic structures, {Mo^VI–M^I–Mo^VI} ↔ {Mo^V–M^III–Mo^V}

SERR spectra of co-immobilized <i>Mhcd</i>₁ and cyt <i>c</i>₅₅₂.

<p><b>A)</b> Ag // 6-mercapto-1-hexanol/1-hexanethiol <b>//</b> cyt <i>c</i>_<b>552</b> // <i>Mhcd</i>_<b>1</b> and <b>B)</b> Ag // 11-amino-1-undecanethiol hydrochloride/1-undecanethiol <b>//</b><i>Mhcd</i>_<b>1</b> // cyt <i>c</i>_<b>552</b> constructs at different poised potentials: 300 mV (green), 200 mV (black) and 0 mV (red). <b>Inset:</b> component analysis of experimental spectra (black traces) in ν_<b>4</b> region of co-adsorbed <i>Mhcd</i>_<b>1</b> and cyt <i>c</i>_<b>552</b> measured at 200 mV; cyt <i>c</i>_<b>552</b> (red) and <i>Mhcd</i>_<b>1</b> (green) populations; overall fit (black). Solid traces designate ferric and dotted traces ferrous ν_<b>4</b> components. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 1.5 mW and 30 s, respectively.</p

Redox titrations of <i>Mhcd</i>₁ in immobilized and solution state.

<p><b>A)</b> SERR spectroelectrochemical titration of <i>Mhcd</i>_<b>1</b> adsorbed on 11-amino-1-undecanethiol hydrochloride/1-undecanethiol coated electrodes. Data points correspond to the relative intensities of ferrous (ν_<b>4</b> at 1362 cm^-1; solid circles) and ferric (ν_<b>4</b> at 1372 cm^-1; open circles) heme <i>c</i> populations, as a function of the electrode potential. Solid lines represent fits of the Nernst equation, <i>E°</i>´ ~ 70 mV, <i>z</i> = 0.44, to the experimental data points. <b>B)</b> RR redox titration of <i>Mhcd</i>_<b>1</b> in solution. The relative intensities of the reduced population are represented as a function of the solution potential, solid circles. The Nernst equation was fitted to the data (black line) with <i>E°</i>´ = 220 ± 5 mV, <i>z</i> = 0.90. <b>Inset:</b> ν_<b>4</b> band of RR spectra measured at solution potentials of (a) 90, (b) 180, (c) 225 and (d) 335 mV. Component spectra represent ferrous (red) and ferric (green) ν_<b>4</b> populations and overall fit (black). The spectra were recorded with 413 nm excitation, with 2 − 3 mW laser power and 40 s accumulation time. <b>Note:</b> Sample preparation for solution RR titrations was performed in anaerobic conditions (glove box). Upon each addition of the reductant, the RR cell was removed from the glove box and the spectra were measured; a fresh aliquot of protein was used for each data point.</p

Determination of the Active Form of the Tetranuclear Copper Sulfur Cluster in Nitrous Oxide Reductase

N₂OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS Cu_Z* form and the 4Cu2S Cu_Z form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the Cu_Z* site accesses the 1-hole and fully reduced redox states, while the Cu_Z site accesses the 2-hole and 1-hole redox states. Single-turnover reactions of N₂OR for Cu_Z and Cu_Z* poised in these redox states and steady-state turnover assays with different proportions of Cu_Z and Cu_Z* show that only fully reduced Cu_Z* is catalytically competent in rapid turnover with N₂O

Potentiometric titration of immobilized cyt <i>c</i>₅₅₂.

<p>SERR spectra of cyt <i>c</i>_<b>552</b> immobilized on 6-mercapto-1-hexanol/1-hexanethiol SAM-coated Ag electrode recorded at electrode potentials of (a) to (d) 250, 300, 350 and 450 mV. All spectra were measured with 413 nm excitation; laser power and accumulation time were 1.5 mW and 30 s, respectively. <b>Inset:</b> relative concentration of ferrous protein (squares) plotted as a function of the electrode potential. The solid line represents a fit of the experimental data to the Nernst equation, yielding <i>E°</i>´ = 262 ± 5 mV, <i>z</i> = 0.70 ± 0.02.</p

RR and SERR spectra of cyt <i>c</i>₅₅₂ and <i>Mhcd</i>₁.

<p><b>A)</b> cyt <i>c</i>_<b>552</b>: RR spectra of (a) ferric and (c) sodium ascorbate reduced, ferrous protein; SERR spectra of cyt <i>c</i>_<b>552</b> immobilized on 6-mercapto-1-hexanol/1-hexanethiol SAM at electrode potentials of (b) 400 mV and (d) −100 mV. <b>B)</b><i>Mhcd</i>_<b>1</b>: RR spectra of (a) ferric and (c) sodium ascorbate reduced, ferrous enzyme; SERR spectra of <i>Mhcd</i>_<b>1</b> on 11-amino-1-undecanethiol hydrochloride/1-undecanethiol SAM at electrode potentials of (b) 300 mV and (d) −300 mV. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 2 − 3 mW and 40 s (RR) or 1.5 − 2.5 mW and 30 s (SERR).</p

RR and SERR spectra of <i>Mhcd</i>₁.

<p>Component analysis of the ν_<b>4</b> region of: RR spectra of <b>A)</b> ferric and <b>C)</b> ferrous <i>Mhcd</i>_<b>1</b> and SERR spectra of <i>Mhcd</i>_<b>1</b> immobilized on a 11-amino-1-undecanethiol hydrochloride/1-undecanethiol SAM at electrode potentials of <b>B)</b> 300 mV and <b>D)</b> −300 mV; green and red solid lines represent native ferric and ferrous populations, respectively, blue line accounts for non-native populations; gray line for non-assigned bands and black line for the overall fit. Red line in panel A indicates traces of photo-reduced protein. <b>E)</b> SERR spectra of <i>Mhcd</i>_<b>1</b> recorded as a function of the electrode potential, from (a) to (e) −150, −50, 50, 100 and 300 mV. The spectra were recorded with 413 nm excitation; laser power and accumulation time were 3 mW and 40 s (RR) or 2.5 mW and 30 s (SERR), respectively.</p

Unusual Reduction Mechanism of Copper in Cysteine-Rich Environment

Copper–cysteine interactions play an important role in Biology and herein we used the copper-substituted rubredoxin (Cu-Rd) from <i>Desulfovibrio gigas</i> to gain further insights into the copper-cysteine redox chemistry. EPR spectroscopy results are consistent with Cu-Rd harboring a Cu^II center in a sulfur-rich coordination, in a distorted tetrahedral structure (<i>g</i>_∥,⊥ = 2.183 and 2.032 and <i>A</i>_∥,⊥ = 76.4 × 10^–4 and 12 × 10^–4 cm^–1). In Cu-Rd, two oxidation states at Cu-center (Cu^II and Cu^I) are associated with Cys oxidation–reduction, alternating in the redox cycle, as pointed by electrochemical studies that suggest internal geometry rearrangements associated with the electron transfer processes. The midpoint potential of [Cu^I(S–Cys)₂(Cys–S–S–Cys)]/[Cu^II(S–Cys)₄] redox couple was found to be −0.15 V vs NHE showing a large separation of cathodic and anodic peaks potential (Δ<i>E</i>_p = 0.575 V). Interestingly, sulfur-rich Cu^II-Rd is highly stable under argon in dark conditions, which is thermodynamically unfavorable to Cu–thiol autoreduction. The reduction of copper and concomitant oxidation of Cys can both undergo two possible pathways: oxidative as well as photochemical. Under O₂, Cu^II plays the role of the electron carrier from one Cys to O₂ followed by internal geometry rearrangement at the Cu site, which facilitates reduction at Cu-center to yield Cu^I(S–Cys)₂(Cys–S–S–Cys). Photoinduced (irradiated at λ_ex = 280 nm) reduction of the Cu^II center is observed by UV–visible photolysis (above 300 nm all bands disappeared) and tryptophan fluorescence (∼335 nm peak enhanced) experiments. In both pathways, geometry reorganization plays an important role in copper reduction yielding an energetically compatible donor–acceptor system. This model system provides unusual stability and redox chemistry rather than the universal Cu–thiol auto redox chemistry in cysteine-rich copper complexes

Spectroscopic Definition of the Cu_Z° Intermediate in Turnover of Nitrous Oxide Reductase and Molecular Insight into the Catalytic Mechanism

Spectroscopic methods and density functional theory (DFT) calculations are used to determine the geometric and electronic structure of Cu_Z°, an intermediate form of the Cu₄S active site of nitrous oxide reductase (N₂OR) that is observed in single turnover of fully reduced N₂OR with N₂O. Electron paramagnetic resonance (EPR), absorption, and magnetic circular dichroism (MCD) spectroscopies show that Cu_Z° is a 1-hole (i.e., 3Cu^ICu^II) state with spin density delocalized evenly over Cu_I and Cu_IV. Resonance Raman spectroscopy shows two Cu–S vibrations at 425 and 413 cm^–1, the latter with a −3 cm^–1 O¹⁸ solvent isotope shift. DFT calculations correlated to these spectral features show that Cu_Z° has a terminal hydroxide ligand coordinated to Cu_IV, stabilized by a hydrogen bond to a nearby lysine residue. Cu_Z° can be reduced via electron transfer from Cu_A using a physiologically relevant reductant. We obtain a lower limit on the rate of this intramolecular electron transfer (IET) that is >10⁴ faster than the unobserved IET in the resting state, showing that Cu_Z° is the catalytically relevant oxidized form of N₂OR. Terminal hydroxide coordination to Cu_IV in the Cu_Z° intermediate yields insight into the nature of N₂O binding and reduction, specifying a molecular mechanism in which N₂O coordinates in a μ-1,3 fashion to the fully reduced state, with hydrogen bonding from Lys397, and two electrons are transferred from the fully reduced μ₄S^2– bridged tetranuclear copper cluster to N₂O via a single Cu atom to accomplish N–O bond cleavage