Modulation of Heme Orientation and Binding by a Single Residue in Catalase HPII of Escherichia coli

Abstract

Heme-containing catalases have been extensively studied, revealing the roles of many residues, the existence of two heme orientations, flipped 180° relative to one another along the propionate-vinyl axis, and the presence of both heme b and heme d. The focus of this report is a residue, situated adjacent to the vinyl groups of the heme at the entrance of the lateral channel, with an unusual main chain geometry that is conserved in all catalase structures so far determined. In Escherichia coli catalase HPII, the residue is Ile274, and replacing it with Gly, Ala, and Val, found at the same location in other catalases, results in a reduction in catalytic efficiency, a reduced intensity of the Soret absorbance band, and a mixture of heme orientations and species. The reduced turnover rates and higher H(2)O(2) concentrations required to attain equivalent reaction velocities are explained in terms of less efficient containment of substrate H(2)O(2) in the heme cavity arising from easier escape through the more open entrance to the lateral channel created by the smaller side chains of Gly and Ala. Inserting a Cys at position 274 resulted in the heme being covalently linked to the protein through a Cys-vinyl bond that is hypersensitive to X-ray irradiation being largely degraded within seconds of exposure to the X-ray beam. Two heme orientations, flipped along the propionate-vinyl axis, are found in the Ala, Val, and Cys variants.

Systematic analysis of residual density suggests that a major limitation in well‐refined X‐ray structures of proteins is the omission of ordered solvent

“…This model is one of the most complete protein models in the PDB examined so far (Table I), and has the smallest R-factor gap with R Ratio of 1.22 (see Methods). [4][5][6][7][8][9][10][11][12][13][14][15] Evidence will be provided below that its corresponding residual ED map is the closest to true featurelessness with all the residual peaks contributed mainly by random noise present in the intensity data.…”

“…17 A few are selected here for this analysis ( Table I). [4][5][6][7][8][9][10][11][12][13][14][15] It is shown that the 5BV2 model 4 appears to be the only one whose intensity differences between the model and data are truly limited by random noise present in the diffraction data (see below).…”

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase-like reactivity ofBacillus pumiluscatalase monitored by X-ray crystallography and EPR spectroscopy

“…Surprisingly, the heme is present with equal proportions of two orientations flipped relative to one another. All clade 3 enzymes so far characterized have one orientation of heme while a clade 1 enzyme (CatF of Pseudomonas syringae ) has predominantly the flipped orientation similar to that in the large subunit KatE from E. coli . It is perhaps relevant that changing the residues adjacent to the heme in KatE did influence the heme orientation leading to mixtures .…”

Role of Active Site Residues on Catalytic Activity of Catalase with Oxidase Activity from Scytalidium Thermophilum

“…Only five different amino acids are found at the location among all catalases, including Val (62%), Ser (32%), Gly (3%), Ile (2%), and Ala (1%) (Jha et al, 2011). To investigate the integrity of this residue in the catalytic function of the enzyme, it was mutated to residues found at the same location in other catalases.…”

“…Thus, the bulky valine or isoleucine restricts movement of H 2 O 2 out of the cavity via the lateral channel, and the longer occupancy in the cavity increases its chances of participating in the reaction. Conversely, opening the lateral channel (with smaller side chains at residue 228) facilitates diffusion of H 2 O 2 out of the cavity, leading to a shorter residency time, poorer substrate binding, and a slower reaction (Jha et al, 2011).…”