Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

Abstract

Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.

Pathways and Mechanisms for Product Release in the Engineered Haloalkane Dehalogenases Explored Using Classical and Random Acceleration Molecular Dynamics Simulations

“…DCL moves through a pathway taking advantage of temporarily increased local space due to natural protein breathing motions, which may be further enhanced by a DCL molecule. This is in agreement with studies describing the accessibility of pathways that are being controlled locally by (i) hydrogen-bonding and salt link interactions, described for cytochrome P450 14,16,[43][44][45][59][60][61][62] and acetylcholinesterase, 63,64 and (ii) aromatic gating, described for cytochrome P450, 14,16,42,43 acetylcholinesterase, 4,64-67 NADH oxidase, 19 horseradish peroxidase, 68,69 and myoglobin. 70 …”

Graphene‐Oxide‐Based Enzyme Nanoarchitectonics for Substrate Channeling

“…Simulation studies indicate that H 2 O 2 enters the heme site with a fluctuating entry point with hydrophobic residues at the surface, resulting in opening and closing of the pore entrance for substrate binding . Upon exposure to a hydrophobic surface, conformational changes make the entry site more accessible, thereby resulting in activity change.…”

Inactivation and Heme Degradation of Horseradish Peroxidase Induced by Discharge Plasma

“…In order to ascertain the origin of the fluorescent product, several control experiments were conducted. Apo‐HRP obtained by removing heme with methyl ethyl ketone in acidic condition at 4 °C were also treated by discharge plasma under the same conditions applied for HRP. The sample shows much weaker fluorescence intensity at 450 nm compared with the HRP sample after plasma treatment (Figure S1 in Supporting Information).…”