6U87

Pseudomonas aeruginosa HasA mutant - Y75H

Summary for 6U87

• Entry DOI: 10.2210/pdb6u87/pdb
• Descriptor: HasAp, PROTOPORPHYRIN IX CONTAINING FE (3 entities in total)
• Functional Keywords: hasa, hemophore, metal binding protein
• Biological source: Pseudomonas aeruginosa
• Total number of polymer chains: 1
• Total formula weight: 21506.28

Authors

Brimberry, M.,Lanzilotta, W.,Wilks, A.,Dent, A. (deposition date: 2019-09-04, release date: 2020-09-09, Last modification date: 2023-10-11)

Primary citation

Dent, A.T.,Brimberry, M.,Albert, T.,Lanzilotta, W.N.,Moenne-Loccoz, P.,Wilks, A.
Axial Heme Coordination by the Tyr-His Motif in the Extracellular Hemophore HasAp Is Critical for the Release of Heme to the HasR Receptor of Pseudomonas aeruginosa .
Biochemistry, 60:2549-2559, 2021

PubMed Abstract: senses extracellular heme via an extra cytoplasmic function σ factor that is activated upon interaction of the hemophore holo-HasAp with the HasR receptor. Herein, we show Y75H holo-HasAp interacts with HasR but is unable to release heme for signaling and uptake. To understand this inhibition, we undertook a spectroscopic characterization of Y75H holo-HasAp by resonance Raman (RR), electron paramagnetic resonance (EPR), and X-ray crystallography. The RR spectra are consistent with a mixed six-coordinate high-spin (6cHS), six-coordinate low-spin (6cLS) heme configuration and an HO exchangeable Fe-O stretching frequency with O/O and H/D isotope shifts that support a two-body Fe-OH oscillator with (iron-hydroxy)-like character as both hydrogen atoms are engaged in short hydrogen bond interactions with protein side chains. Further support comes from the EPR spectrum of Y75H holo-HasAp that shows a LS rhombic signal with ligand-field splitting values intermediate between those of His-hydroxy and bis-His ferric hemes. The crystal structure of Y75H holo-HasAp confirmed the coordinated solvent molecule hydrogen bonded through H75 and H83. The long-range conformational rearrangement of HasAp upon heme binding can still take place in Y75H holo-HasAp, because the intercalation of a hydroxy ligand between the heme iron and H75 allows the variant to reproduce the heme binding pocket observed in wild-type holo-HasAp. However, in the absence of a covalent linkage to the Y75 loop combined with the malleability provided by the bracketing H75 and H83 hydrogen bonds, either the hydroxy sixth ligand remains bound after complexation of Y75H holo-HasAp with HasR or rearrangement and coordination of H85 prevent heme transfer.

PubMed: 34324310
DOI: 10.1021/acs.biochem.1c00389

Experimental method

X-RAY DIFFRACTION (1.3 Å)