Finding Gas Diffusion Pathways in Proteins: Application to O2 and H2 Transport in CpI [FeFe]-Hydrogenase and the Role of Packing Defects

Abstract

We report on a computational investigation of the passive transport of H2 and O2 between the external solution and the hydrogen-producing active site of CpI [FeFe]-hydrogenase from Clostridium pasteurianum. Two distinct methodologies for studying gas access are discussed and applied: (1) temperature-controlled locally enhanced sampling, and (2) volumetric solvent accessibility maps, providing consistent results. Both methodologies confirm the existence and function of a previously hypothesized pathway and reveal a second major pathway that had not been detected by previous analyses of CpI's static crystal structure. Our results suggest that small hydrophobic molecules, such as H2 and O2, diffusing inside CpI, take advantage of well-defined preexisting packing defects that are not always apparent from the protein's static structure, but that can be predicted from the protein's dynamical motion. Finally, we describe two contrasting modes of intraprotein transport for H2 and O2, which in our model are differentiated only by their size.

Experimental approaches to kinetics of gas diffusion in hydrogenase

“…However, these theoretical results have not been confronted with experimental observations, because methods for determining the rates of diffusion inside proteins are lacking. It is not always established whether small diatomic molecules transit through static tunnels (6) or dynamic pathways (12), and to the best of our knowledge, there is only one reported example of a structurally characterized mutant of an enzyme showing impeded intramolecular diffusion (15).…”

“…This approach unveils the role of conformational flexibility and questions the requirement for a static channel to transport small diatomic molecules. In the case of the FeFe hydrogenase mentioned above, such simulations revealed a ''dynamic'' channel that had not been detected as a cavity in the frozen crystal (12). Whether the channel is static or dynamically formed, it may consist of a single or multiple routes for gas transport.…”

“…Their affinity for gas molecules was sometimes confirmed by showing that they can bind Xenon in the crystal (10,11). Alternatively, molecular dynamics (MD) calculations can evaluate the dynamics of gas permeation through the protein matrix (11)(12)(13)(14). This approach unveils the role of conformational flexibility and questions the requirement for a static channel to transport small diatomic molecules.…”

Exploring Molecular Oxygen Pathways in Hansenula polymorpha Copper-containing Amine Oxidase

“…Trajectories were then recorded for 10-ns simulations performed at constant pressure (1 atm, Langevin piston) and temperature (300 K, Langevin dynamics) using long-range PME electrostatics and the CHARMM22 force-field (43) with custom parameters built by analogy for the TPQ residue and histidine-copper bonds. The trajectories were used as input for an implicit ligand sampling analysis (44) contained in the VMD software package (45), resulting in detailed three-dimensional free energy profiles for O 2 placement inside the protein based on the assumption that the presence of gas molecules can be treated as a weak perturbation to protein natural thermal motion (46).…”

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

“…[19][20][21][22] Oxygen diffusion in myoglobin's distal pocket has been extensively studied, both experimentally and by simulations, in light of the influence of different protein conformations or mutations. 14,23 In FPs, the interaction between the chromophore and the surrounding protein has important implications for both structures.…”