Dynamics of carbon monoxide binding to CooA.
CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.
Department of Chemistry, Princeton University, Frick Laboratory, Washington Road, Princeton, NJ 08544, USA.
Mrinalini
M
Puranik
Steen Brøndsted
SB
Nielsen
Hwan
H
Youn
Angela N
AN
Hvitved
James L
JL
Bourassa
Martin A
MA
Case
Charbel
C
Tengroth
Gurusamy
G
Balakrishnan
Marc V
MV
Thorsteinsson
John T
JT
Groves
George L
GL
McLendon
Gary P
GP
Roberts
John S
JS
Olson
Thomas G
TG
Spiro
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012247:Rhodospirillum rubrum / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D015534:Trans-Activators; D002248:Carbon Monoxide