Abstract
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 k app1 = 9.6±0.2 M–1 s–1 and k app2 = 1.2±0.1 M–1 s–1 (at pH 7.4 and 20°C). The k app1 and k app2 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 h app = 3.8×104 M–1 s–1 and h 0 = 2.8×10–1 s–1 (at pH 7.4 and 20°C). The pH-dependence of h on and h 0 values reflects the acid-base equilibrium of peroxynitrite (pK a = 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.
Highlights
Phylogenetic analysis revealed that members of the globin superfamily evolved from an ancestral monomeric flavo-hemoglobin and were arranged in three globin lineages and two structural classes
All members of the first and third lineage belong to the same structural class, showing a long amino acid sequence (.145 residues) and the classical 3-on-3 a-helical fold found in myoglobin, with the heme group surrounded by the A, B, and E a-helices on one side and the F, G, and H a-helices on the other [3,4]
Methanosarcina acetivorans Pgb (Ma-Pgb) shows a selectivity ratio for O2/CO binding that favors O2 ligation [11] and displays anti-cooperativity in CO binding [15]. This very unusual behavior could be related to the fact that Methanosarcina acetivorans takes advantage of acetate, methanol, CO2 and CO as carbon sources for methanogenesis; methane production occurs simultaneously with the formation of a proton gradient that is essential for energy harvesting [9,52,53]
Summary
Phylogenetic analysis revealed that members of the globin superfamily evolved from an ancestral monomeric flavo-hemoglobin and were arranged in three globin lineages and two structural classes. All members of the first and third lineage belong to the same structural class, showing a long amino acid sequence (.145 residues) and the classical 3-on-3 a-helical fold found in myoglobin, with the heme group surrounded by the A, B, and E a-helices on one side and the F, G, and H a-helices on the other [3,4]. All members of the second lineage, consisting of three subgroups, belong to the same structural class and show a short amino acid sequence (,135 residues), adopting the 2-on-2 a-helical sandwich fold, characterized by a very short or absent A-helix, a brief CE inter-helical region and most of the F-helix occurring as a loop, with only the B, E, G, and H a-helices surrounding the heme [5]. The access of ligands to the heme distal pocket is granted by two protein matrix tunnels, which are located at the B/G (tunnel 1) and B/E (tunnel 2) a-helix interfaces (Fig. 1, panel B) [11,12,13,14,15,16]
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