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J. Biol. Chem., Vol. 279, Issue 20, 21096-21108, May 14, 2004

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Dynamics of Carbon Monoxide Binding to CooA^*

From the ^aDepartment of Chemistry, Princeton University, Princeton, New Jersey 08544, the ^dDepartment of Bacteriology, University of Wisconsin at Madison, Madison, Wisconsin 53706, and the ^eDepartment of Biochemistry and Cell Biology, Rice University, Houston, Texas 77025

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.

Received for publication, January 20, 2004 , and in revised form, February 25, 2004.

^* This work was supported in part by National Institutes of Health (NIH) Grants GM33576 (to T. G. S.), GM53228 (to G. P. R.), GM36928 (to J. T. G.), GM 35649 (to J. S. O.), HL 47020 (to J. S. O.), by National Science Foundation Grant CHE 99-09502 (to M. A. C. and G. L. M.), and by Grant C-612 (to J. S. O.) from the Robert A. Welch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^b Both authors contributed equally to this work.

^c Supported by a postdoctoral fellowship from the Danish Natural Science Research Council (Grant 21-01-0142). Current address: Dept. of Physics and Astronomy, University of Aarhus, Ny Munkegade, DK-8000 Aarhus C, Denmark.

^f Recipient of a traineeship from the NIH Training Grant GM08280.

^g Supported by Grant CHE-0106342 from the National Science Foundation Division of Chemistry.

^h Current address: Bioprocess Research and Development, Merck Research Laboratories, West Point, PA 19486.

i To whom correspondence may be addressed: Dept. of Biochemistry and Cell Biology, Rice University, MS 140, 6100 Main St., Houston, TX 77025-1892. Tel.: 713-348-4762; Fax: 713-348-5154; E-mail: olson{at}rice.edu. j To whom correspondence may be addressed: Dept. of Chemistry, Princeton University, Frick Laboratory, Washington Rd., Princeton, NJ 08544. Tel.: 609-258-3907; Fax: 609-258-0348; E-mail: spiro{at}princeton.edu.

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