TY - JOUR
T1 - Dynamics of carbon monoxide binding to CooA
AU - Puranik, Mrinalini
AU - Nielsen, Steen Brøndsted
AU - Youn, Hwan
AU - Hvitved, Angela N.
AU - Bourassa, James L.
AU - Case, Martin A.
AU - Tengroth, Charbel
AU - Balakrishnan, Gurusamy
AU - Thorsteinsson, Marc V.
AU - Groves, John Taylor
AU - McLendon, George L.
AU - Roberts, Gary P.
AU - Olson, John S.
AU - Spiro, Thomas G.
PY - 2004/5/14
Y1 - 2004/5/14
N2 - 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, P50 = 2.2 μM, and estimated Adair constants K1 = 0.16 and K2 = 1.3 μM-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 (≥1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.
AB - 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, P50 = 2.2 μM, and estimated Adair constants K1 = 0.16 and K2 = 1.3 μM-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 (≥1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.
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U2 - 10.1074/jbc.M400613200
DO - 10.1074/jbc.M400613200
M3 - Article
C2 - 14990568
AN - SCOPUS:2442647919
SN - 0021-9258
VL - 279
SP - 21096
EP - 21108
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 20
ER -