Electronic Journal of Biology

Keywords

Aqueous solutions; Molybdenumcontaining- enzymes; Malic acid; Mo⁶⁺; Nitrogenase; Potentiometry.

Introduction

Among all micronutrients, Molybdenum possesses very unique characters. It is the only second row transition metal that has a tangible biological activity, it exists in a wide variety of oxidation states (ranging from 0 to +6) and it is a required co-factor for at least four dozen enzymes [1]. There are a limited number of reviews or mini-reviews that have appeared with the biology/biochemistry of molybdenum in mind [1- 3], particularly in aqueous solutions. However, the meticulous and thorough 75 page review by Hille et al. [2] is a great reference for the biochemistry of molybdenum of which they cited 536 other biomolybdenum- related research articles.

Herein, we have conducted detailed literature research to prepare for this min-review and found the following three facts: (1) Not many research articles dealt with the reaction of Mo⁶⁺ and aqueous solutions; (2) The chemistry of molybdenum is extremely complex; and (3) Billions of years ago, nature understood the uniqueness of molybdenum biochemistry that scientists only recently have recognized [1-20]. Figure 1 of the supplementary materials shows the details of this library search of the molybdenum research articles and review articles that were found within all American Chemical Society (ACS) Journals. It is noteworthy that there are a total of 44 journals within all ACS publication domains, which publishes thousands of research papers and reviews monthly.

Figure 1: Potentiometric titration graph of free Mo6+ in aqueous solutions, 25°C, I=0.10 M (NaNO₃). Six replicas show data presission.

There are at least four dozens known molybdenumcontaining- enzymes and or proteins (molybdoenzymes); Nitrogenase being the most well-known among all of them within the biology and chemistry audience. Herein, we are going to mention a dozen as example of these known molybdo-enzymes: (1) Nitrogenase, (2) Nitrate Reductase, (3) Xanthine Oxidase or Xanthine Dehydrogenase, (4) Pyrimidine Oxidase/Aldehyde Oxidase, (5) Trimethylamine Oxide Reductase, (6) Formate Dehydrogenase (7) Carbon Monoxide Oxoreductase/Carbon Monoxide Dehydrogenase, (8) Pyridoxal Oxidase, (9) Sulfite Oxidase, (10) Biotin Sulfoxide Reductase, (11) Dimethyl Sulfoxide Redutase, and (12) Tetrathionite Reductase. Table 1 catalogues all of these enzymes. Some of these Molybdenum-containingenzymes were isolated from bacteria (particularly cyanobacteria), fungi, yeast, plants, or mammals. For more details refer to references 1-3 and all 694 references mentioned therein. This current mini-review will focus on the discussion of the Molybdenum-containing-enzymes “Nitrogenase”.

Table 1: Enzyme commission numbers and functions of all molybdenum containing enzymes mentioned in this mini-review.

Nitrogenase, isolated from N₂-fixing bacteria, is the enzyme catalyzing the reduction of molecular N₂ to ammonia (NH₃). In addition to the reduction of molecular nitrogen N₂, Nitrogenase can also reduce a variety of small, unsaturated substrates including the reduction of C₂H₂ to C₂H₄, N₂O to N₂ and H₂O, N₃^- to NH₄⁺ and N₂, and HCN to CH₃NH₂ [17,18]. Nitrogenase is composed of two proteins, dinitrogenase (FeMo protein) and dinitrogenase reductase (Fe protein). Dinitrogenase reductase is a dimer of two identical α₂ subunits that bridge one ferredoxin-like (Fe₄S₄)^2+/1+ cluster. Dinitrogenase is a α₂β₂ tetramer, and it carries a unique iron-molybdenum cluster that contains Fe, Mo and S with one cagelike MoFe7S9 homocitrate cluster per α, β-pair. It is believed that the substrate molecules (such as molecular nitrogen, N₂) bind inside this FeMo cluster [4-8]. The Mo center is octahedrally coordinated to three sulfides (μ3-S ligands), a histidine imidazole side chain, and two oxygen atoms from homocitrate. Supplementary Figure 2 shows the structure formula of the active site of the FeMo cofactor in the N₂ fixing enzyme; Nitrogenase.

Figure 2: Number of proton equivalent versus slopes for the six free Mo⁶⁺ potentiometric titration.

Burris and coworkers demonstrated that citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumonia [9]. In dinitrogenase, the homocitrate ligand is coordinated to the Mo center via the central carboxyl and the central hydroxyl group. We have visited this subject a decade ago with our detailed speciation article [19]. Molybdenum is believed to be taken up by organisms as the molybdate anion MoO₄^2-. This would be essential for the biosynthesis of the final FeMo cofactor from an oxomolybdenum citrate precursor [13-16]. Tricarboxylic acids, citric and homocitric acids may play an essential role in the mobilization of molybdenum during cofactor biosynthesis and the mobilized oxomolybdenum tricarboxylate fragment must undergo reduction, exchange the oxo ligands for the sulfide ligands, and merge with the rest of the nifB. While the precise role of tricarboxylic acids in the biosynthesis of the FeMo cofactor and the mechanism of dinitrogenase reduction is still regarded as poorly understood [15], the elucidation of the role played by the tricarboxylic acids has been pursued with great interest [9-16,19].

Many researchers, including ourselves, have attempted to mimic the reaction of Mo⁶⁺ with that of mono-hydroxyl poly-carboxylates (citrate, homocitrate and malate) [9- 16,19-23]. Herein, we show the outcome of the reactions of the simple mono-hydroxyl di-carboxylate (Malate) in the form of the protonated Malic acid (H₂MA) with Mo⁶⁺ in aqueous solutions under ambient conditions in 0.1 M ionic strength (NaNO₃).

Experimental Section

Materials and method

Aqueous solutions of D,L-Malic acid (H₂MA) were prepared using 99% purity (Sigma reagent grade), formula weight 134.09 g.mol^-1. Aqueous molybdenum (Mo⁶⁺) solutions were prepared from the crystalline ammonium salt of molybdic acid, (NH₄)6Mo7O₂4.4H₂O, 83% MoO₃, formula weight 1235.9 g.mol^-1, also Sigma reagent grade. The primary (1°) standard potassium hydrogen phthalate (KHP, 99.99%) and the solid sodium hydroxide pellets (NaOH, 98%) were purchased from Fisher Chemical Company. We have standardized the sodium hydroxide (NaOH) solution to the fourth digits to the right of the decimal point. All pH values were measured using Thermo-Orion Membrane Advanced ISE/pH/mV/ORP meter (model 720A+) connected to the accurate-combination Orion-glass electrode in 0.1 mol.L^-1 ionic strength. The 0.10 M ionic strengths of all solutions were adjusted by the addition of 10% v/v of 1.0 M NaNO₃ solution.

Potentiometry

The detailed methods used to carry potentiometric titrations are well established in the literature [19,21- 24]. For each individual experiment, H₂MA solutions were first added to the titration vessel, followed by the addition of the specified amount of Mo⁶⁺ solution. The final total volume of the reacting species was 100 mL. NaOH solution was added in 100 μL increments by means of the calibrated and accurate, 100 μL total capacities, Eppendorf micro-pipette. Each individual potentiometric titration took about 3.5 h to complete.

Results and Discussion

Figure 1 shows the potentiometric titration plots of free Mo⁶⁺. The free H₂MA titration curve has been published in previous studies [24,25-27]. It is established that H₂MA releases two protons out of the two carboxylic acid groups. Additionally, the hydroxyl group can participate in metal ion chelation to form two fused six-membered and five-membered very robust ring chelates [19,25-27]. Figure 2 shows the potentiometric titration plots of Mo⁶⁺ in the form of number of proton equivalents released (x-axis) versus the first derivatives or slopes (y-axis) of the experimental pH-values (the inflection points appeared at 7.78 ± 0.10, n=6). This observation parallels that of the previous study of Mo⁶⁺ with citric acid which is consistent with the literature values [19].

Figure 3 is the double plots of free H₂MA in which the potentiometric titrations of six replicas in the form of volume of added titrant versus experimental pH-values are overlaid with the number of proton equivalents on the x-axis versus the slopes on the y-axis. A similar titration plateau was observed in our previous studies [25,27]. This particular titration graph is the very same graph that has been collected alongside the reaction of the hexavalent molybdenum (Mo⁶⁺) in its reaction with malic acid (H₂MA); see below for details of the Mo⁶⁺ reaction with H₂MA.

Figure 3: Potentiometric titration graph of six replicas of free Malic Acid in aqueous solutions, 25°C, I=0.1 M (NaNO₃)

Figure 4 shows the titration plateaus of the reaction mixture of Mo⁶⁺ with MA in 1:3 M ratios. Excess amount of malic acid was selected for three reasons: (1) to avoid metal ion hydrolysis; (2) to avoid the formation of any oligomeric metal-complexes that were formed when the ratio of the Mo⁶⁺ ion was in excess [21-23]; and (3) for ease of pipetting 2.00 mL H₂MA with 2.00 mL Mo⁶⁺, which gave this 1:3 molar reaction ratios. After calculating the number of proton equivalents of the reactions of Mo⁶⁺ with H₂MA in 1:3 M ratios (Figure 5), it appeared that ~ 17 proton equivalents were released into the solution (17.01 ± 1.22, n=6). This observation is in a very good agreement that one might expect for the release of a net 7.78 proton equivalents from the free mole of Mo⁶⁺ in addition to the net of nine protons total out of the three available protons to be released per malic acid molecule (i.e., 9H⁺ from the three moles of H₂MA plus 7.78 from the free Mo⁶⁺ ion). In n Figure 5, it shows the values which are equal to 16.78 proton equivalents.

Figure 4: Potentiometric titration of Mo⁶⁺ : Malic acid in 1:3 ratios. 25 °C, I =0.10 M (NaNO₃)

Figure 5: Number of H⁺ equivalent versus slopes of the Mo6+: Malic acid titrations in 1:3 ratio shown in Figure 4. More than 17H⁺ were released due to the binding of Mo⁶⁺ to MA

Figure 5 confirms that the malic acid is releasing three H⁺ equivalents although it is referred to as H₂MA in the literature [19,24-27]. This is due to the central hydroxyl group upon chelation to Mo⁶⁺ (or any metal ion for that matter) releases its proton. It is concluded from this data that the main complexes that have been formed in solution are those according to equation 1.

Conclusion

The current experimental data presented in this mini-review is novel. When the detailed literature reviews were searched for the reactions of H₂MA with the hexavalent molybdenum ion Mo⁶⁺, only few papers were found [21-23]. In these studies, they all agreed that the aqueous solution chemistry of Mo⁶⁺ is not that simple. They also agreed on the formation of the simple one to one complexes [MoO₂(MA) (OH)₂]^3-, [MoO₂(MA)₂]^4-, [MO₂O₅(MA)2(OH)₂]^4- with stability constant values of Logβ of 8.17, 13.89 and 22.31, respectively [22]. When the crystal structure was published two years later after these initial aqueous attempts in 1983 [21], it turned out that what crystallized was the bis-mono-protonated complex which has the following molybdenum malate complex formula: [MoO₂(Hmal)₂]^2- [21]. We do not believe that we are observing this bis-mono-protonated complex. What concerns us here is not whether we are observing the formation of the di- anion, the tri-anion, or the tetra-anion molybdenum-malate complex. We are also not concerned with whether it is the monoprotonated or the de-protonated or the (hydroxo) molybdenum-malate complex. However, in this study the significant revelation is that we have identified the total number of proton equivalents released into the solution which gives the same binding mode as seen in the crystal structure and the previous equilibrium studies [21-24].

The detailed and the most trusted reviews of the molybdoenzymes by researchers [1-3] stressed that the bi-dentate binding mode of the monohydroxyl poly-carboxylates, whether it is in the form of citrate or homocitrate, is the dominant mode of binding. Here in, we are stressing that the formed complex of Mo⁶⁺ with malic acid released a net of 17 proton equivalents which can only be accounted for by the binding of Malate in a bi-dentate or a tridentate fashion (the potentiometric titration is lacking supplying this information). The stoichiometry given in the equilibrium shown in equation (1) is only accounted for by the formation of a mixture of the two proposed molybdenum-malate complexes depicted, i.e., [MoO₂(H-1MA)(OH)₂]^3- + [MoO₂(H-1MA)₂]^4-. This mixture of the two complexes released a net of 17 H⁺. It is noteworthy that these two complexes are consistent with the ones identified by others [21-24].

Acknowledgement

We would like to acknowledge the financial support from NSF under Grant # HRD-1332459. Special thanks go to Professor Sherry Painter for reading the manuscript and her many helpful suggestions. of awards and prizes.

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