The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

Abstract

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, solved at sub-Ångstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To understand the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or the tunnel residues Phe359 or Gly347 to Trp or Asn, reduces the catalytic efficiency of oxidation. The K mO2 increases from 300 ± 35 μM for the wild-type enzyme to 617 ± 15 μM for the F359W mutant. The k cat for the F359W mutant catalyzed reaction decreases 13-fold relative to the wild-type catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Hydride transfer from the sterol to the flavin prosthetic group is no longer rate limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel-mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively are consistent with the cooperative kinetics. In the atomic resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is only observed in a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

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).…”

“…The mutations strongly increase the Michaelis constant for H 2 , with no appreciable effect on the value of k cat (extrapolated to infinite concentration of H 2 in Table 1). This phenotype is expected when a mutation slows substrate access to the active site (4,5,8,15).…”

“…In a few cases, however, mutations of some residues located in a putative gas channel did affect the enzyme's kinetic properties. For example, certain mutations in enzymes that use molecular oxygen as substrate increase the K m for O 2 (4,5,8,15). Brzezinski and coworkers (6) demonstrated that a glycine-to-valine mutation almost completely obstructs the oxygen channel of cytochrome c oxidase; in this study, the delayed access of substrate O 2 and inhibitor CO to the active-site heme was probed by using time-resolved UV-vis spectroscopy.…”

“…Several alanine mutations were also found to block the long channel in ACS-CODH (2, 3). However, the structure of the mutants has been determined in only one of the above cases (15). This structural information is important, because mutations may have unexpected structural consequences; for example, a glycine to phenylalanine mutation in the channel of carbamoyl-phosphate synthetase causes a large conformational change that creates an escape route for ammonia directly to the bulk solvent (16).…”

A comparative analysis of clustering algorithms: O2 migration in truncated hemoglobin I from transition networks

“…[32][33][34] More recent work found that O 2 follows specific tunnels (see above), [35][36][37] may involve multiple pathways, and is likely to be coupled (slaved) to the protein motion. [38][39][40][41][42] The possibility for a ligand to follow multiple pathways was, for example, demonstrated experimentally and through MD simulation for CO in Mb and O 2 -migration in flavoenzymes.…”