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

PLoS One. 2015 Jun 5;10(6):e0125959. doi: 10.1371/journal.pone.0125959. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Binding Sites
  • Carbon Monoxide / chemistry
  • Carbon Monoxide / metabolism*
  • Crystallography, X-Ray
  • Hydrogen Bonding
  • Kinetics
  • Ligands
  • Methanosarcina / metabolism*
  • Mutagenesis, Site-Directed
  • Photolysis
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Spectrophotometry, Ultraviolet
  • Spectrum Analysis, Raman

Substances

  • Archaeal Proteins
  • Ligands
  • Recombinant Proteins
  • Carbon Monoxide

Grants and funding

The authors acknowledge the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2008, 2008BFJ34R, and Azioni Integrate Italia Spagna 2009, IT10L1M59M), and Ministero degli Affari Esteri, Direzione generale per la promozione del sistema Paese (Progetti di Grande Rilevanza, Italia-Argentina 2011-2013), the University of Antwerp and the Fund for Scientific Research, Flanders, Belgium (Grant N° G.0247.09) for financial support. This work was partially supported by grants from the Ministry for University and Research of Italy (University Roma Tre, Roma, Italy, “CLAR 2012”) and by institutional funds from University of Milano (FIRST-2007 and PUR-2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.