Production of the Carbonate Radical Anion during Xanthine Oxidase Turnover in the Presence of Bicarbonate

Abstract

Xanthine oxidase is generally recognized as a key enzyme in purine catabolism, but its structural complexity, low substrate specificity, and specialized tissue distribution suggest other functions that remain to be fully identified. The potential of xanthine oxidase to generate superoxide radical anion, hydrogen peroxide, and peroxynitrite has been extensively explored in pathophysiological contexts. Here we demonstrate that xanthine oxidase turnover at physiological pH produces a strong one-electron oxidant, the carbonate radical anion. The radical was shown to be produced from acetaldehyde oxidation by xanthine oxidase in the presence of catalase and bicarbonate on the basis of several lines of evidence such as oxidation of both dihydrorhodamine 123 and 5,5-dimethyl-1-pyrroline-N-oxide and chemiluminescence and isotope labeling/mass spectrometry studies. In the case of xanthine oxidase acting upon xanthine and hypoxanthine as substrates, carbonate radical anion production was also evidenced by the oxidation of 5,5-dimethyl-1-pyrroline-N-oxide and of dihydrorhodamine 123 in the presence of uricase. The results indicated that Fenton chemistry occurring in the bulk solution is not necessary for carbonate radical anion production. Under the conditions employed, the radical was likely to be produced at the enzyme active site by reduction of a peroxymonocarbonate intermediate whose formation and reduction is facilitated by the many xanthine oxidase redox centers. In addition to indicating that the carbonate radical anion may be an important mediator of the pathophysiological effects of xanthine oxidase, the results emphasize the potential of the bicarbonate-carbon dioxide pair as a source of biological oxidants.

Direct Magnetic Resonance Evidence for Peroxymonocarbonate Involvement in the Cu,Zn-Superoxide Dismutase Peroxidase Catalytic Cycle

“…Recent literature has reported several developments that have been important in unraveling the SOD1 peroxidase cycle: first, the demonstration that SOD1 peroxidase activity is greatly stimulated by HCO 3 Ϫ (5, 6) and dependent on CO 2 (8); second, the characterization by Richardson's group (27) . (30,33). This idea has inspired continued investigation (30, 31, 39 -41).…”

“…Although this process is well described for the heme-dependent peroxidase cycle, current literature data (9,(27)(28)(29)(30) and the fact that the proposed SOD1-bound hypervalent copper states (Cu(III), Cu(II)ϭO, and Cu(II)/ ⅐ OH) have never been characterized suggested to us that an alternative mechanism may take place, leading to CO 3 . production from HCO 3 Ϫ and H 2 O 2 by the enzyme, a process that does not involve copper oxidation beyond the thermodynamically stable Cu(II) form.…”

Experimental Studies on the Chemiluminescence Reaction Mechanism of Carbonate/Bicarbonate and Hydrogen Peroxide in the Presence of Cobalt(II)

“…A series of experiments were conducted to establish optimum analytical 4 on the CL intensity was tested further, and the results are shown in Figure 3a. These results revealed the fact that Co 2+ was the limiting reactant.…”

Involvement of carbonate/bicarbonate ions in the superoxide-generating reaction of adrenaline autoxidation

“…То есть конечные продукты реакции автоокисления адреналина, кроме адренохрома, могут быть не только супероксид анионы, но и радикалы карбонат/бикарбонатного буфера и диоксида углерода, растворённого в буфере. Такое наше предположение основывается на данных современной литературы, где активно обсуждается существование и роль карбонатных радикалов и радикалов диоксида углерода [30][31][32][33][34][35][36][37][38]42].…”

“…В известной ферментативной модельной системе ксантин/ксантиноксидаза также генерируются не только супероксид анион радикалы, пероксид водорода и пероксинитрит, но и продуцируются карбонатные анион радикалы, которые, как предполагается, могут быть важными медиаторами в патофизиологических эффектах ксантиноксидазы [36]. В литературе демонстрируются и обсуждаются возможные пути образования при различных условиях пероксимонокарбоната и карбонатных радикалов как патогенетических оксидантов из вездесущего физиологического буфера карбонат/бикарбонат/диоксид углерода [33,37].…”