A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding, signal transduction, and structure.
To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.
Dunham
Christine M
CM
Chemistry and Biochemistry Department, University of California, 236 Sinsheimer Labs, Santa Cruz, California 95064, USA.
Dioum
Elhadji M
EM
Tuckerman
Jason R
JR
Gonzalez
Gonzalo
G
Scott
William G
WG
Gilles-Gonzalez
Marie-Alda
MA
D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D020369:Bradyrhizobium / Q000378:metabolism; D003460:Crystallization; D006420:Hemeproteins / Q000378:metabolism*; D000071677:Histidine Kinase; D008024:Ligands; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D001120:Arginine; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase