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.
Highlights
Methanosarcina acetivorans (M. acetivorans) is an obligate anaerobic archaeon that was isolated from marine sediments where kelp decomposition, e.g., decomposition to methane, occurs [1]
The available experimental data on the CO binding kinetics of MaPgbÃ(II) show that an equilibrium exists between two tertiary conformations of the liganded Pgb: MaPgbÃr(II)-CO and MaPgbÃt(II)-CO; this equilibrium favors MaPgbÃr(II)-CO over MaPgbÃt(II)-CO [10]
The integrated use of site-directed mutagenesis, X-ray crystallography, vibrational spectroscopy, and ligand-binding kinetics has allowed the identification of the stereochemical mechanisms that form the basis of CO binding and CO sensing and of the alterations in the conformational state of the heme-binding pocket
Summary
Methanosarcina acetivorans (M. acetivorans) is an obligate anaerobic archaeon that was isolated from marine sediments where kelp decomposition, e.g., decomposition to methane, occurs [1]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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