Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO2– to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are kapp1 = 9.6±0.2 M–1 s–1 and kapp2 = 1.2±0.1 M–1 s–1 (at pH 7.4 and 20°C). The kapp1 and kapp2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are happ = 3.8×104 M–1 s–1 and h0 = 2.8×10–1 s–1 (at pH 7.4 and 20°C). The pH-dependence of hon and h0 values reflects the acid-base equilibrium of peroxynitrite (pKa = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.
Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Roma, Italy;National Institute of Biostructures and Biosystems, Roma, Italy;Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Roma, Italy;Department of Physics, University of Genova, Genova, Italy;Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Roma, Italy;Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy;Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Roma, Italy;Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy;Department of Bioscience, University of Milano, Milano, Italy;Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Roma, Italy;Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy
Recommended Citation:
Paolo Ascenzi,Loris Leboffe,Alessandra Pesce,et al. Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin[J]. PLOS ONE,2014-01-01,9(5)