The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O 2 , CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 3 10 helix (H234 on E), stabilizes O 2 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. O 2 appeared to be the native Aer2 ligand (dissociation constant [K d ] of 16 M). With one exception, mutants that bound O 2 could signal, whereas many mutants that bound CO could not. W283 stabilized O 2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O 2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O 2 with wildtype affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O 2 binding, L264A and L264G caused O 2 -mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O 2 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.IMPORTANCE Pseudomonas 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 O 2 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.KEYWORDS chemoreceptor, PAS domain, signal transduction, Pseudomonas aeruginosa, heme, oxygen P seudomonas aeruginosa is a common environmental bacterium and a significant cause of opportunistic human disease. It survives in complex environments with the aid of 26 chemoreceptors and four chemosensory systems that collectively sense environmental conditions and modify bacterial behavior. The roles of three of these chemosensory systems are known: one modulates type IV pili production and twitching motility (Pil-Chp system), another controls biofilm formation (Wsp system), and a third

Summary The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer-PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N-terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase-off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS-HAMP surfaces overlapped with a cluster of PAS kinase-on lesions and with cysteine substitutions that crosslinked the PAS β-scaffold to the HAMP AS-2 helix. A refined Aer PAS-HAMP interaction model is presented. Compared to the kinase-off state, the kinase-on state increased the accessibility of HAMP residues (apparently relaxing PAS-HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static-dynamic model in which oxidized Aer-PAS interacts directly with HAMP AS-2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase-off output. When PAS-FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase-on output.