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

Abstract

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.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study

“…The kinetics of NO 2 − reduction from Mt -Nb(II), At -Nb(II), Dr -Nb(II), and Hs -Nb(II) (i.e., of Nb(II)-NO formation) were analyzed in the framework of the minimum reaction mechanism depicted in Scheme 1 [ 27 , 29 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 53 ].…”

Kinetic and dynamical properties of truncated hemoglobins of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

“…Following the approach we exploited other Hbs, both pentacoordinated, like human Hb (Sottini, Abbruzzetti, Spyrakis, et al, 2005), protoglobin (Tilleman et al, 2015), and hexa-coordinated, like human Cygb (Gabba et al, 2013), Ngb (Abbruzzetti et al, 2009;Boron, Capece, et al, 2015) plant Hbs (Abbruzzetti et al, 2021(Abbruzzetti et al, , 2007Bisht et al, 2011), and nitrophorins (Abbruzzetti, He, et al, 2012), we used a minimal kinetic model, reported in Scheme 1, to perform a global analysis of V 1 amplitudes at 1 and 0.1 atm CO. The scheme, derived from the MD simulations results, takes into account transient binding at the primary docking site, ligand exchange with the solvent, and migration to an off-pathway temporary docking site (Tr2).…”