Ligation Tunes Protein Reactivity in an Ancient Haemoglobin: Kinetic Evidence for an Allosteric Mechanism in Methanosarcina acetivorans Protoglobin

Abbruzzetti, Stefania; Tilleman, Lesley; Bruno, Stefano; Viappiani, Cristiano; Desmet, Filip; Doorslaer, Sabine Van; Coletta, Massimo; Ciaccio, Chiara; Ascenzi, Paolo; Nardini, M.; Bolognesi, Martino; Moëns, Luc; Dewilde, Sylvia

doi:10.1371/journal.pone.0033614

Cited by 14 publications

(31 citation statements)

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“…On the other hand, the 149–154 region might be able to influence/modulate the architecture and ligand binding properties of the haem distal cavity through association of an (unknown) effector molecule or partner protein. Whether such regulation might occur in an allosteric fashion is a further possibility, although (negative) cooperativity has been reported for O 2 but not for CO binding [6], [30].…”

Section: Discussionmentioning

confidence: 98%

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

et al. 2013

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Protoglobin from Methanosarcina acetivorans C2A (MaPgb), a strictly anaerobic methanogenic Archaea, is a dimeric haem-protein whose biological role is still unknown. As other globins, protoglobin can bind O2, CO and NO reversibly in vitro, but it displays specific functional and structural properties within members of the hemoglobin superfamily. CO binding to and dissociation from the haem occurs through biphasic kinetics, which arise from binding to (and dissociation from) two distinct tertiary states in a ligation-dependent equilibrium. From the structural viewpoint, protoglobin-specific loops and a N-terminal extension of 20 residues completely bury the haem within the protein matrix. Thus, access of small ligand molecules to the haem is granted by two apolar tunnels, not common to other globins, which reach the haem distal site from locations at the B/G and B/E helix interfaces. Here, the roles played by residues Trp(60)B9, Tyr(61)B10 and Phe(93)E11 in ligand recognition and stabilization are analyzed, through crystallographic investigations on the ferric protein and on selected mutants. Specifically, protein structures are reported for protoglobin complexes with cyanide, with azide (also in the presence of Xenon), and with more bulky ligands, such as imidazole and nicotinamide. Values of the rate constant for cyanide dissociation from ferric MaPgb-cyanide complexes have been correlated to hydrogen bonds provided by Trp(60)B9 and Tyr(61)B10 that stabilize the haem-Fe(III)-bound cyanide. We show that protoglobin can strikingly reshape, in a ligand-dependent way, the haem distal site, where Phe(93)E11 acts as ligand sensor and controls accessibility to the haem through the tunnel system by modifying the conformation of Trp(60)B9.

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Section: Discussionmentioning

confidence: 98%

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

et al. 2013

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“…This CO concentration allowed to obtain the fully saturated Ma-Pgb*-Fe(II)-CO complex, the value of the overall dissociation equilibrium constant being about 1×10 −8 M [15].…”

Section: Methodsmentioning

confidence: 99%

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

et al. 2014

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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.

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“…The cells were resuspended in 50 mM Tris-HCl pH 8.0, 5.0 mM EDTA, 0.5 mM DTT and 1.0 mM phenylmethylsulphonyl fluoride. The bacterial cells were lysed, and recombinant proteins were purified from inclusion bodies as described previously [ 10 ].…”

Section: Methodsmentioning

confidence: 99%

“…Resonance Raman (RR) spectra of Ma Pgb*(II)-CO mutants were recorded using a Dilor XY-800 Raman scattering spectrometer consisting of a triple 800 mm spectrograph operating in low-dispersion mode and using a liquid nitrogen-cooled CCD detector as described previously [ 10 ]. The excitation source for all spectra was a mixed Ar/Kr laser (Spectra-Physics Beamlok 2060) operating at 413.1 nm.…”

Section: Methodsmentioning

confidence: 99%

“…The equilibrium is shifted towards the species with low binding (and high dissociation) rates for the unliganded molecules. Biphasic CO binding kinetics was observed in flash-photolysis and stopped-flow experiments, showing that ferrous Ma Pgb* r ( Ma Pgb* r (II)) is characterized by a high CO association rate and low CO dissociation rate, whereas ferrous Ma Pgb* t ( Ma Pgb* t (II)) shows a low CO association rate and a high CO dissociation rate [ 10 ]. Although Ma Pgb* assembles into a homodimer in solution, this structure does not result in cooperative CO binding.…”

Section: Introductionmentioning

confidence: 99%

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Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

et al. 2015

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Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.

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Ligation Tunes Protein Reactivity in an Ancient Haemoglobin: Kinetic Evidence for an Allosteric Mechanism in Methanosarcina acetivorans Protoglobin

Cited by 14 publications

References 50 publications

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Structure and Haem-Distal Site Plasticity in Methanosarcina acetivorans Protoglobin

Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

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