Role of Distal Arginine in Early Sensing Intermediates in the Heme Domain of the Oxygen Sensor FixL

• Published in: Biochemistry (Easton) Vol. 45; no. 19; pp. 6018 - 6026
• Main Authors: Jasaitis, Audrius, Hola, Klara, Bouzhir-Sima, Latifa, Lambry, Jean-Christophe, Balland, Veronique, Vos, Marten H, Liebl, Ursula
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 16.05.2006
• Subjects: Arginine - chemistry; Bacterial Proteins - chemistry; Carbon Monoxide - chemistry; Heme - chemistry; Hemeproteins - chemistry; Hydrogen Bonding; Kinetics; Oxygen - chemistry; Spectrum Analysis - methods
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi060012i

FixL is a bacterial heme-based oxygen sensor, in which release of oxygen from the sensing PAS domain leads to activation of an associated kinase domain. Static structural studies have suggested an important role of the conserved residue arginine 220 in signal transmission at the level of the heme domain. To assess the role of this residue in the dynamics and properties of the initial intermediates in ligand release, we have investigated the effects of R220X (X = I, Q, E, H, or A) mutations in the FixLH heme domain on the dynamics and spectral properties of the heme upon photolysis of O2, NO, and CO using femtosecond transient absorption spectroscopy. Comparison of transient spectra for CO and NO dissociation with steady-state spectra indicated less strain on the heme in the ligand dissociation species for all mutants compared to the wild type (WT). For CO and NO, the kinetics were similar to those of the wild type, with the exception of (1) a relatively low yield of picosecond NO rebinding to R220A, presumably related to the increase in the free volume of the heme pocket, and (2) substantial pH-dependent picosecond to nanosecond rebinding of CO to R220H, related to formation of a hydrogen bond between CO and histidine 220. Upon excitation of the complex bound with the physiological sensor ligand O2, a 5−8 ps decay phase and a nondecaying (>4 ns) phase were observed for WT and all mutants. The strong distortion of the spectrum associated with the decay phase in WT is substantially diminished in all mutant proteins, indicating an R220-induced role of the heme in the primary intermediate in signal transmission. Furthermore, the yield of dissociated oxygen after this phase (∼10% in WT) is increased in all mutants, up to almost unity in R220A, indicating a key role of R220 in caging the oxygen near the heme through hydrogen bonding. Molecular dynamics simulations corroborate these findings and suggest motions of O2 and arginine 220 away from the heme pocket as a second step in the signal pathway on the 50 ps time scale.