TY - JOUR
T1 - A Proximal Arginine R206 Participates in Switching of the Bradyrhizobium japonicum FixL Oxygen Sensor
AU - Gilles-Gonzalez, Marie-Alda
AU - Caceres, Ana Isabel
AU - Sousa, Eduardo Henrique Silva
AU - Tomchick, Diana R
AU - Brautigam, Chad A
AU - Gonzalez, Constancio
AU - Machius, Mischa
N1 - Funding Information:
We thank Kelly Krabill-Gerber for preparing mutant genes and Sandra Hill for providing excellent technical assistance. The project was supported by a Welch Foundation grant no. I-1575, by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2002-35318-14039, and by a Spanish DGICYT grants BFI 2001-1713 and BFU 2004-06394. Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Energy Research, under contract no. W-31-109-ENG-38.
PY - 2006/6/30
Y1 - 2006/6/30
N2 - In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd∼350 μM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the FeIII form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.
AB - In oxygen-sensing PAS domains, a conserved polar residue on the proximal side of the heme cofactor, usually arginine or histidine, interacts alternately with the protein in the "on-state" or the heme edge in the "off-state" but does not contact the bound ligand directly. We assessed the contributions of this residue in Bradyrhizobium japonicum FixL by determining the effects of an R206A substitution on the heme-PAS structure, ligand affinity, and regulatory capacity. The crystal structures of the unliganded forms of the R206A and wild-type BjFixL heme-PAS domains were similar, except for a more ruffled porphyrin ring in R206A BjFixL and a relaxation of the H214 residue and heme propionate 7 due to their lost interactions. The oxygen affinity of R206A BjFixL (Kd∼350 μM) was 2.5 times lower than that of BjFixL, and this was due to a higher off-rate constant for the R206A variant. The enzymatic activities of the unliganded "on-state" forms, either deoxy or met-R206A BjFixL, were comparable to each other and slightly lower (twofold less) than those of the corresponding BjFixL species. The most striking difference between the two proteins was in the enzymatic activities of the liganded "off-state" forms. In particular, saturation with a regulatory ligand (the FeIII form with cyanide) caused a >2000-fold inhibition of the BjFixL phosphorylation of BjFixJ, but a 140-fold inhibition of this catalytic activity in R206A BjFixL. Thus, in oxygen-sensing PAS domains, the interactions of polar residues with the heme edge couple the heme-binding domain to a transmitter during signal transduction.
KW - FixJ
KW - PAS domain
KW - response regulator
KW - sensor kinase
KW - two-component system
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U2 - 10.1016/j.jmb.2006.04.054
DO - 10.1016/j.jmb.2006.04.054
M3 - Article
C2 - 16813836
AN - SCOPUS:33745240395
SN - 0022-2836
VL - 360
SP - 80
EP - 89
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -