The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra-and extracellular compartments. Peroxynitrite anion (ONOO ؊ ) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO 2 ؊ ), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO 3 . ). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6 -9. The radical was unambiguously identified by its EPR parameters (g ؍ 2.0113; line width ؍ 5.5 G) and by experiments with bicarbonate labeled with 13 C. In this case, the singlet EPR signal obtained with 12 C bicarbonate splits into the expected doublet because of 13 C (a( 13 C)؍ 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO 3 . ).Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.Peroxynitrite 1 is formed from the very fast reaction between nitric oxide and superoxide anion (k ϭ (6.7 Ϫ 19) ϫ 10 9 M Ϫ1 s Ϫ1 ) (see Reaction 1) (1, 2). The compound is a potent oxidant that has been receiving increasing attention as a potential pathogenic mediator in human diseases and as a cellular toxin in host defense mechanisms against invading microorganisms (3-6). At present, a significant part of the biological reactivity of peroxynitrite is ascribed to the adduct produced by its reaction with carbon dioxide (7-13). The peroxynitrite anion (ONOO Ϫ ), which is the predominant form at physiological pHs (pK a ϭ 6.8) (see reaction 2, Table II) (2, 3), reacts fast with carbon dioxide (pH-independent k ϭ 5.8 ϫ 10 4 M Ϫ1 ⅐ s Ϫ1 at 37°C) (11), producing an adduct whose structure is proposed to be ([ONOOCO 2 ] Ϫ , nitrosoperoxocarboxylate) (see reaction 3, Table II) (7). Taking into account the concentrations of carbon dioxide in equilibrium with bicarbonate present in physiological fluids, model calculations have suggested that most of the peroxynitrite that might be formed in these fluids will produce the carbon dioxide adduct before reacting with other biological targets (5, 13).Carbon dioxide modulates the reactivity of peroxynitrite by altering reaction rates, product yields, and product distribution (7-13). In these reactions, formation of the adduct nitrosoperoxocarboxylate is rate-limiting, as first proposed by Lymar and Hurst (7). This suggestion was confirmed by other authors (8 -13), and the current proposal is that in the absence of substrates, the carbon dioxide adduct decomposes to nitrate and carbon dioxide, but in ...
Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.
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