Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins

Abstract

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c-NO and 6c-CO complexes but do not form O 2 complexes. Structure of the AxCYTcp-CO complex at 1.25 Å resolution shows that CO binds in two conformations moderated by the extent of displacement of the distal residue Leu16 toward the haem 7-propionate. The presence of two CO conformations is confirmed by cryogenic resonance Raman data. The preferred linear Fe-C-O arrangement (170 AE 8°) is accompanied by a flip of the propionate from the distal to proximal face of the haem. In the second conformation, the Fe-C-O unit is bent (158 AE 8°) with no flip of propionate. The energetic cost of the CO-induced Leupropionate movements is reflected in a 600 mV (57.9 kJmol −1 ) decrease in haem potential, a value in good agreement with density functional theory calculations. Substitution of Leu by Ala or Gly (structures determined at 1.03 and 1.04 Å resolutions) resulted in a haem site that binds CO in the linear mode only and where no significant change in redox potential is observed. Remarkably, these variants were isolated as ferrous 6c-CO complexes, attributable to the observed eight orders of magnitude increase in affinity for CO, including an approximately 10,000-fold decrease in the rate of dissociation. These new findings have wide implications for preventing CO poisoning of gas-binding haem proteins.sensors | crystallography | kinetics | ligand discrimination | atomic resolution

Folding helical proteins in explicit solvent using dihedral-biased tempering

“…In fact, the majority [e.g., cytochrome ć (PDB ID: 3ZTM) (32) Additional results of the simulated proteins are discussed in the SI Text and Figs. S11-S17.…”

Modulation of ligand–heme reactivity by binding pocket residues demonstrated in cytochrome c' over the femtosecond–second temporal range

“…Previous studies have shown that L16A binds NO on the distal face in a 6c‐NO mode (with the proximal histidine still attached) 12, without proceeding to a proximal binding mode. Consequently, this variant of cytochrome c' is a useful comparative system.…”

“…The heme environment in crystal structures of (A) ferrous A x C ytcp at 1.45 Å resolution ( PDB ID 2YLI) 12 and (B) ferrous, NO ‐bound A x C ytcp at 1.2 Å resolution ( PDB ID 2XLM) 13, with key binding‐pocket residues shown.…”

“…The concentration of the ferrous sample was estimated using ε 426  = 97 000  m −1  cm −1 31. The L16A variant was isolated with CO bound to the heme distal face as previously reported 12. This ligand was removed by incubating with an excess of potassium ferricyanide (500 m m ) for 1 h at room temperature under anaerobic conditions.…”

The pH Dependent Protein Structure Transitions and Related Spin-State Transition of Cytochrome c′ from Alcaligenes xylosoxidans NCIMB 11015

“…1723 The active site structure of the CO and NO bound Cyt c¤ was changed, with the orientation of the several amino-acids side chains in the binding site region altered by the presence of the exogenous ligand binding. 18,23 While the exact physiological function of the Cyt c¤ has not been elucidated, it has been suggested that Cyt c¤ may be an NO-reservoir or electron-transfer agent. The reported reduction potential of Cyt c¤ from Alcaligenes xylosoxidans (AxCyt c¤) is 184 mV vs. NHE, 16 which is lower than the reduction potential for the general cytochrome c.…”