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

Nitrite Reductase Activity of Rat and Human Xanthine Oxidase, Xanthine Dehydrogenase, and Aldehyde Oxidase: Evaluation of Their Contribution to NO Formation <i>in Vivo</i>

Nitrite is presently considered a NO “storage form” that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme’s specificity constant for nitrite increase 8-fold, while the <i>K</i>_m^{NO₂^–} decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the <i>K</i>_i^O₂ values found (24.3–48.8 μM) suggest that <i>in vivo</i> NO formation would be fine-tuned by dioxygen availability. The potential <i>in vivo</i> relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised

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}

Gd(III) Chelates as NMR Probes of Protein–Protein Interactions. Case Study: Rubredoxin and Cytochrome <i>c</i>₃

Two cyclen-derived Gd probes, [Gd–DOTAM]³⁺ and [Gd–DOTP]^5– (DOTAM = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide; DOTP = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), were assessed as paramagnetic relaxation enhancement (PRE)-inducing probes for characterization of protein–protein interactions. Two proteins, <i>Desulfovibrio gigas</i> rubredoxin and <i>Desulfovibrio gigas</i> cytochrome <i>c</i>₃, were used as model partners. In a ¹H NMR titration it was shown that [Gd–DOTP]^5– binds to cytochrome <i>c</i>₃ near heme IV, causing pronounced PREs, characterized by line width broadenings of the heme methyl resonances at ratios as low as 0.08. A<i> K</i>_d of 23 ± 1 μM was calculated based on chemical shift perturbation of selected heme methyl resonances belonging to three different heme groups, caused by allosteric effects upon [Gd–DOTP]^5– binding to cytochrome <i>c</i>₃ at a molar ratio of 2. The other probe, [Gd–DOTAM]³⁺, caused PREs on a well-defined patch near the metal center of rubredoxin (especially the patch constituted by residues D19–G23 and W37–S45, which broaden beyond detection). This effect was partially reversed for some resonances (C6–Y11, in particular) when cytochrome <i>c</i>₃ was added to this system. Both probes were successful in causing reversible PREs at the partner binding site, thus showing to be good probes to identify partners’ binding sites and since the interaction is reversible to structurally characterize protein complexes by better defining the complex interface

The Sulfur Shift: An Activation Mechanism for Periplasmic Nitrate Reductase and Formate Dehydrogenase

A structural rearrangement known as sulfur shift occurs in some Mo-containing enzymes of the DMSO reductase family. This mechanism is characterized by the displacement of a coordinating cysteine thiol (or SeCys in Fdh) from the first to the second shell of the Mo-coordination sphere metal. The hexa-coordinated Mo ion found in the as-isolated state cannot bind directly any exogenous ligand (substrate or inhibitors), while the penta-coordinated ion, attained upon sulfur shift, has a free binding site for direct coordination of the substrate. This rearrangement provides an efficient mechanism to keep a constant coordination number throughout an entire catalytic pathway. This mechanism is very similar to the carboxylate shift observed in Zn-dependent enzymes, and it has been recently detected by experimental means. In the present paper, we calculated the geometries and energies involved in the sulfur-shift mechanism using QM-methods (M06/(6-311++G­(3df,2pd),SDD)//B3LYP/(6-31G­(d),SDD)). The results indicated that the sulfur-shift mechanism provides an efficient way to enable the metal ion for substrate coordination

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

One Electron Reduced Square Planar Bis(benzene-1,2-dithiolato) Copper Dianionic Complex and Redox Switch by O₂/HO^–

The complex [Ph₄P]₂[Cu­(bdt)₂] (<b>1</b>^<b>red</b>) was synthesized by the reaction of [Ph₄P]₂[S₂MoS₂CuCl] with H₂bdt (bdt = benzene-1,2-dithiolate) in basic medium. <b>1</b>^<b>red</b> is highly susceptible toward dioxygen, affording the one electron oxidized diamagnetic compound [Ph₄P]­[Cu­(bdt)₂] (<b>1</b>^<b>ox</b>). The interconversion between these two oxidation states can be switched by addition of O₂ or base (Et₄NOH = tetraethylammonium hydroxide), as demonstrated by cyclic voltammetry and UV–visible and EPR spectroscopies. Thiomolybdates, in free or complex forms with copper ions, play an important role in the stability of <b>1</b>^<b>red</b> during its synthesis, since in its absence, <b>1</b>^<b>ox</b> is isolated. Both <b>1</b>^<b>red</b> and <b>1</b>^<b>ox</b> were structurally characterized by X-ray crystallography. EPR experiments showed that <b>1</b>^<b>red</b> is a Cu­(II)–sulfur complex and revealed strong covalency on the copper–sulfur bonds. DFT calculations confirmed the spin density delocalization over the four sulfur atoms (76%) and copper (24%) atom, suggesting that <b>1</b>^<b>red</b> has a “thiyl radical character”. Time dependent DFT calculations identified such ligand to ligand charge transfer transitions. Accordingly, <b>1</b>^<b>red</b> is better described by the two isoelectronic structures [Cu^I(bdt₂, 4S^3–,*)]^2– ↔ [Cu^II(bdt₂, 4S^4–)]^2–. On thermodynamic grounds, oxidation of <b>1</b>^<b>red</b> (doublet state) leads to <b>1</b>^<b>ox</b> singlet state, [Cu^III(bdt₂, 4S^4–)]^1–