Pulse Radiolysis Studies on the Reaction of the Reduced Vitamin B12 Complex Cob(II)alamin with Superoxide

Reactive oxygen and nitrogen species (ROS/RNS) including hydroxyl radicals, nitric oxide, hydrogen peroxide, peroxynitrite and superoxide, are important signaling molecules and are also generated at high concentrations as part of the innate immune response to pathogens.[1] However, elevated levels of reactive species can lead to oxidative stress, resulting in irreversible damage to biomolecules.[1] Oxidative stress has been implicated in numerous diseases including cardiovascular disease, cancer, neurological diseases and aging in general.[1-2] This has led to considerable interest in the development of therapeutics that scavenge ROS/RNS, including porphyrin-based complexes.[3] The structurally related vitamin B12derivatives cobalamins (Cbls; X = Me or Ado, Scheme 1), are cofactors for two enzyme reactions in humans – cytosolic methionine synthase (MS), and mitochondrial L-methylmalonyl-CoA mutase (MM-CoA mutase).[4] Cbl supplementation has been shown to be beneficial for treating chronic inflammatory diseases including chronic fatigue syndrome, sepsis, asthma, rheumatoid arthritis, neurological diseases including Alzheimer’s disease, and viral based diseases including AIDS.[5] Recently Scalabrino et al demonstrated that Cbl attenuates the pro-inflammatory immune response in a rat model for chronic Cbl deficiency by modulating levels of cytokines and growth factors including TNF-α, nerve growth factor, epidermal growth factor and IL-6.[6] Scheme 1 Structure of vitamin B12derivatives (cobalamins). X = CH3, Ado, H2O/OH, NO, etc. The important ROS superoxide, O2·−, is produced as a byproduct of cellular metabolism by mitochondrial and reticular membrane electron transport systems and by enzymes such as NADPH oxidase and xanthine oxidase.[7] O2·− is a precursor of two of the most potent ROS/RNS, peroxynitrite and the hydroxyl radical.[8] Superoxide dismutases, enzymes that catalyze the destruction of O2·− by converting it to O2 and H2O2, are upregulated in oxidative stress - associated diseases.[1] In vitro cell studies using human aortic endothelial cells show that vitamin B12prevents O2·− - induced oxidative stress at physiologically relevant (~10−9M) concentrations.[9] With supplementation intracellular Cbl levels can reach 0.7 μM.[10] Upon entering cells vitamin B12derivatives (cob(III)alamins) are reduced by the cblC protein to cob(II)alamin, Cbl(II).[11] We have previously shown that O2·− generated by the xanthine oxidase/acetaldehyde system reacts rapidly with the radical complex Cbl(II) by an indirect competition method.[9b] Given the importance of this reaction in human health, we now report the direct determination of rate constants for the reaction between Cbl(II) and O2·− using pulse radiolysis to generate O2·−. Figure 1 gives a plot of absorbance at 530 nm versus time for the reaction between Cbl(II) (7.3 × 10−5M) and O2·− (9.0 × 10−6M) at pH 7.42. Irradiating aqueous solutions generates ·OH, eaq− and ·H,[12] and in O2-saturated aqueous solutions containing ethanol or formate, these primary radicals are rapidly converted to O2·−.[12] An anaerobic Cbl(II) solution was rapidly mixed with an O2- saturated aqueous ethanol solution using a stopped-flow mixing device. The resulting mixture was subsequently exposed to an intense ionizing pulse (30 – 300 ns) and data collected. The time between mixing and pulsing was typically <2 s and the Cbl(II) concentration was kept at least 5 times higher than the O2·− concentration, to achieve essentially pseudo-first order conditions. Our earlier stoichiometry experiments showed that the reaction between Cbl(II) and O2·− proceeds according to eq (1), with clean oxidation of the metal center of Cbl(II) to give aquacobalamin/hydroxycobalamin, H2OCbl+/HOCbl (pKa(H2OCbl+) = 7.8[13]).[9b] Cbl(II)+O2⋅−+2H3O+→H2OCbl++H2O2+H2O (1) Figure 1 Plot of change in absorbance at 530 nm versus time for the reaction of O2·− (9.0 × 10−6M) with excess Cbl(II) (7.3 × 10−5M) at pH 7.42 (0.500 M EtOH, 5.00×10−3M phosphate buffer, I = 0.010 ... The data in Figure 1 fit well to a first-order rate equation, giving an observed rate constant, kobs= (2.70 ± 0.02) × 104s−1. The rate constant is unchanged when data are collected at 435 nm (kobs = (2.69 ± 0.05) × 104s−1; Figure S1, Supporting Information). Rate constants for the reaction between Cbl(II) and O2·− were also determined at other Cbl(II) concentrations at pH 7.42 and the data are summarized in Figure 2. The data fit well to a straight line passing through the origin, consistent with a single irreversible reaction. The linear relationship suggests that the reaction is first-order with respect to Cbl(II) and O2·−. From the slope, the second-order rate constant (kapp) of the reaction was (3.78 ± 0.07) × 108M−1s−1. Separate pulse radiolysis experiments showed that H2OCbl+/HOCbl does not react with O2·− (pH 7.42, 0.500 M EtOH, I = 0.01 M (phosphate buffer)). Analyzing this latter data at 260 nm gave a rate constant for O2·− decomposition similar to that reported in the literature at this pH value (decomposition of superoxide is pH dependent).[12] Values of kappwere also determined at pH 5.50 and 8.72 at a single Cbl(II) concentration, giving kapp = (4.23 ± 0.18) × 108 M−1s−1 and (4.40 ± 0.22) × 108M−1s−1, respectively (Figures S2 and S3, Supporting Information). Given the similarity in these rate constants to the value obtained at pH 7.42 (3.78 ± 0.07) × 108M−1 s−1), kappis essentially independent of pH in this pH range. Figure 2 Plot of kobsversus Cbl(II) concentration for the reaction between excess Cbl(II) ((1.6 - 7.3) × 10−5M) and O2·− ((1.0 – 9.0) × 10−6M) at pH 7.42 (0.500 M EtOH, 5.00×10−3M phosphate ... It is well established that Cbl(II) is oxidized to H2OCbl+/HOCbl by air.[9b] In order to confirm that Cbl(II) is also oxidized to H2OCbl+/HOCbl by O2·− in our pulse radiolysis experiments, a plot of change in extinction coefficient versus wavelength for the reaction of Cbl(II) with O2·− was generated and compared with the change in extinction coefficient for the Cbl(II)/(H2OCbl+/HOCbl) conversion, Figure 3. (Note that it was not possible to obtain full spectra as a function of time from our experimental setup for the pulse radiolysis experiments.) There is excellent agreement between the two sets of data and the isosbestic points observed at 375, 490 and 578 nm correspond to the expected values reported for the Cbl(II)/(H2OCbl+/HOCbl) conversion.[9b] The corresponding plots of absorbance of the reaction product solution versus wavelength for both sets of data are also in excellent agreement with each other (Figure S4, Supporting Information). The formation of H2OCbl+/HOCbl from the reaction between Cbl(II) and O2·− was additionally confirmed by HPLC analysis of the product solution from the pulse radiolysis experiments and by using the xanthine oxidase/acetaldehyde system to generate O2·− (details given in the Supporting Information). Figure 3 Change in the molar absorption coefficient versus wavelength for the reaction of (■) excess Cbl(II) ((1.6 – 7.3) × 10−5M) with O2·− ((1.0 – 9.0) × 10−6M) at pH 7.42 (0.500 M EtOH, ... The amount of H2O2 produced in the reaction between Cbl(II) and O2·−, eq (1), was found to be ~0.96 mol equiv. using the H2O2-specific Amplex Red assay from a calibration plot of fluorescence intensity of oxidized Amplex Red versus H2O2 concentration.[14]O2·− was generated using the xanthine oxidase/acetaldehyde system for these experiments. Details are given in the Supporting Information. To summarize, the rate constant of the reaction between Cbl(II) by O2·− has been directly determined using pulse radiolysis and was found to be 3.8 × 108M−1s−1(pH 7.42, RT). This value is in reasonable agreement with our earlier rate constant value (7.0 × 108M−1s−1, 0.010 M phosphate buffer, pH 7.40, 25 °C),[9a] which was determined indirectly using a competitor and generating O2·− using the xanthine oxidase/acetaldehyde system. Cbl(II) is cleanly oxidized to H2OCbl+/HOCbl and the rate of the reaction is independent of pH in the pH 5.52-8.72 region, as expected, since neither Cbl(II) nor O2·− ionize in this region. The rate constant for the reaction between Cbl(II) and O2·− is within an order of magnitude as that reported for cytosolic Cu/Zn superoxide dismutase (Cu/Zn SOD; 2.0 × 109M−1s−1, pH 5.0-9.5, 25.0 °C[15]) and mitochondrial manganese superoxide dismutase (MnSOD; 1.1 × 109M−1s−1pH 8.2, RT[16]). Cob(II)alamin is a major intracellular cobalamin form and both intracellular cobalamin and SOD concentrations can reach micromolar levels.[11b, 17] Intracellular reductases subsequently reduce H2OCbl+/HOCbl to Cbl(II).[11]Cell studies also show that Cbl prevents O2·− - induced oxidative stress.[9] Given these latter results combined with the similarity of intracellular concentrations of Cbl and SOD and the rate constants for the reactions of SOD or Cbl(II) with O2·−, this supports a role for Cbl as a O2·− scavenger in addition to acting as a cofactor for the two mammalian B12-dependent enzyme reactions. Since a significant proportion (up to 30-40%) of the elderly population are vitamin B12 deficient[18] and vitamin B12is non-toxic even at high doses,[18]B12 supplementation may be especially beneficial for treating chronic inflammatory diseases associated with aging. Enzyme-bound Cbl(II) is also likely to be rapidly oxidized to its inactive H2OCbl+/HOCbl form by O2·−, consistent with inactivation of the B12enzymes under oxidative stress conditions.[19]