Insights into the signal transduction mechanism of RmFixL provided by carbon monoxide recombination kinetics.
This report presents evidence for interdomain steps of the ligand-coupled signal transduction mechanism of the oxygen receptor from Rhizobium meliloti, RmFixL. Photolysis of the CO adducts of heme domain (RmFixLN) and heme kinase (RmFixL*) proteins allowed tracking of second-order heme CO recombination reactions by transient absorbance. Whereas CO rebinding to RmFixLN is characterized by a single kinetic phase, rebinding to RmFixL* is characterized by two kinetic phases. Evidence indicates that CO rebinds to two interconvertible deoxyRmFixL* conformers that are produced sequentially after photolysis. Since the second conformer is only observed when the kinase domain is present, its production is concluded to be an interdomain signal transmission event that is coupled to heme ligand release. Because receptor clustering is a recurring theme in signal transduction mechanisms, the dependence of molecular weight upon heme ligation was investigated at equilibrium. Gel permeation chromatography and native gel electrophoresis showed that the molecular weight distribution for both RmFixLN and RmFixL* depends on heme ligation. At equilibrium, oxyRmFixLN and oxyRmFixL* exist as monomers and dimers, respectively. Their deoxy analogues, metRmFixLN and metRmFixL*, exist as dimers and as a mixture of tetramers and 9-mers, respectively. Assembly of these oligomers is reversible. The physiological relevance of these ligand-coupled assemblies and the kinetic factors controlling CO recombination are discussed.
Rodgers
K R
KR
Department of Chemistry, North Dakota State University, Fargo, North Dakota 58105, USA. kent_rodgers@ndsu.nodak.edu
Tang
L
L
Lukat-Rodgers
G S
GS
Wengenack
N L
NL
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D008024:Ligands; D008027:Light; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D006418:Heme; D002248:Carbon Monoxide; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen