ABSTRACT: The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 310 helix (H234 on E?), stabilizes O2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [Kd ] of 16 ?M). With one exception, mutants that bound O2 could signal, whereas many mutants that bound CO could not. W283 stabilized O2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O2 with wild-type affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O2 binding, L264A and L264G caused O2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response.IMPORTANCEPseudomonas aeruginosa is a ubiquitous environmental bacterium and opportunistic pathogen that infects multiple body sites, including the lungs of cystic fibrosis patients. P. aeruginosa senses and responds to its environment via four chemosensory systems. Three of these systems regulate biofilm formation, twitching motility, and chemotaxis. The role of the fourth system, Che2, is unclear but has been implicated in virulence. The Che2 system contains a chemoreceptor called Aer2, which contains a PAS sensing domain that binds heme and senses oxygen. Here, we show that Aer2 uses unprecedented mechanisms to bind O2 and initiate signaling. These studies provide both the first functional corroboration of the Aer2-PAS signaling mechanism previously proposed from structure as well as a signaling model for Aer2-PAS receptors.
