Role of conserved F(alpha)-helix residues in the native fold and stability of the kinase-inhibited oxy state of the oxygen-sensing FixL protein from Sinorhizobium meliloti.
The oxygen-sensing FixL protein from Sinorhizobium meliloti is part of the heme-PAS family of gas sensors that regulate many important signal transduction pathways in a wide variety of organisms. We examined the role of the conserved F(alpha)-9 arginine 200 and several other conserved residues on the proximal F(alpha)-helix in the heme domain of SmFixL* using site-directed mutagenesis in conjunction with UV-visible, EPR, and resonance Raman spectroscopy. The F(alpha)-helix variants R200A, E, Q, H, Y197A, and D195A were expressed at reasonable levels and purified to homogeneity. The R200I and Y201A variants did not express in observable quantities. Tyrosine 201 is crucial for forming the native protein fold of SmFixL* while Y197 and R200 are important for stabilizing the kinase-inhibited oxy state. Our results show a clear correlation between H-bond donor ability of the F(alpha)-9 side chain and the rate of heme autoxidation. This trend in conjunction with crystal structures of liganded BjFixL heme domains, show that H-bonding between the conserved F(alpha)-9 arginine and the heme-6-propionate group contributes to the kinetic stability of the kinase-inactivated, oxy state of SmFixL*.
Reynolds
Mark F
MF
Department of Chemistry, Saint Joseph's University, 5600 City Avenue, Philadelphia, PA 19131, USA. mreynold@sju.edu
Ackley
Lindsey
L
Blizman
Alice
A
Lutz
Zachary
Z
Manoff
David
D
Miles
Matthew
M
Pace
Matthew
M
Patterson
Joseph
J
Pozzessere
Nicholas
N
Saia
Kathryn
K
Sato
Risa
R
Smith
Danielle
D
Tarves
Paul
P
Weaver
Matthew
M
Sieg
Kristina
K
Lukat-Rodgers
Gudrun S
GS
Rodgers
Kenton R
KR
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D017510:Protein Folding; D016962:Sinorhizobium meliloti / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods
D001426:Bacterial Proteins; D006420:Hemeproteins; D010770:Phosphotransferases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen