The Asp99–Arg188 salt bridge of the Pseudomonas aeruginosa HemO is critical in allowing conformational flexibility during catalysis

Abstract

The P. aeruginosa iron-regulated heme oxygenase (HemO) is required within the host for the utilization of heme as an iron source. As iron is essential for survival and virulence, HemO represents a novel antimicrobial target. We recently characterized small molecule inhibitors that bind to an allosteric site distant from the heme pocket, and further proposed binding at this site disrupts a nearby salt bridge between D99 and R188. Herein, through a combination of site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry (HDX-MS), we determined that the disruption of the D99-R188 salt bridge leads to significant decrease in conformational flexibility within the distal and proximal helices that form the heme-binding site. The RR spectra of the resting state Fe(III) and reduced Fe(II)-deoxy heme-HemO D99A, R188A and D99/R188A complexes are virtually identical to those of wild-type HemO, indicating no significant change in the heme environment. Furthermore, mutation of D99 or R188 leads to a modest decrease in the stability of the Fe(II)-O heme complex. Despite this slight difference in Fe(II)-O stability, we observe complete loss of enzymatic activity. We conclude the loss of activity is a result of decreased conformational flexibility in helices previously shown to be critical in accommodating variation in the distal ligand and the resulting chemical intermediates generated during catalysis. Furthermore, this newly identified allosteric binding site on HemO represents a novel alternative drug-design strategy to that of competitive inhibition at the active site or via direct coordination of ligands to the heme iron.

Alison Butler: papers in celebration of her 2018 ACS Alfred Bader Award in Bioorganic or Bioinorganic Chemistry

“…Turning to heme enzymes, Wilks and co-authors [15] report that disruption of an aspartic acid/arginine salt bridge in HemO, the heme oxygenase from Pseudomonas aeruginosa, leads to complete loss of enzymic activity. Bren, Elliott and co-authors [16] report on the heme ruffling and electronic structure in covalently attached heme in different motifs, for example, with three of four intervening residues, as in CX 3 CH or CX 4 CH, in variants of Hydrogenobacter thermophilus cytochrome c552.…”

Gallium(III)–Salophen as a Dual Inhibitor of Pseudomonas aeruginosa Heme Sensing and Iron Acquisition

“…Differences in the deuterium uptake over time are reflective of how readily backbone amide hydrogens of a protein exchange with the solvent hydrogens or deuterons in a manner that is dependent on the structure, solvent-exposure, and flexibility. We have previously used this technique to describe ligand-induced structural and dynamic changes of the heme-binding proteins PhuS and HemO. , Here, heme binding resulted in significant protection from deuteration throughout the heme binding site, consistent with loop closure and decreased solvent exposure (Figure C). Similar protection of the heme site was observed upon GaSal binding, further suggesting that GaSal binds to HasAp in the heme site and is coordinated with the same ligands (Figure D).…”

“…We have previously used this technique to describe ligand-induced structural and dynamic changes of the heme-binding proteins PhuS and HemO. 52,53 Here, heme binding resulted in significant protection from deuteration throughout the heme binding site, consistent with loop closure and decreased solvent exposure (Figure 5C). Similar protection of the heme site was observed upon GaSal binding, further suggesting that GaSal binds to HasAp in the heme site and is coordinated with the same ligands (Figure 5D).…”

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase

“…Five of the perturbed residues (L20, L21, F48, F117, and V118) were found within the heme binding site of HemO on the proximal (helices I and II) or distal helices (helices VII and VIII) as seen on the HemO crystal structure (Figure 6C), of which F117 has been shown to directly interact with the heme, specifically the methyl and vinyl groups. 30,55 The other three residues (A163, E169, and E174) are located at the edge of the heme pocket on helices XI and XII. The perturbation of these residues is likely due to conformational changes in the proximal and distal helices upon binding of the ligand.…”