Dynamics of carbon monoxide binding to CooA

Mrinalini Puranik, Steen Brøndsted Nielsen, Hwan Youn, Angela N. Hvitved, James L. Bourassa, Martin A. Case, Charbel Tengroth, Gurusamy Balakrishnan, Marc V. Thorsteinsson, John Taylor Groves, George L. McLendon, Gary P. Roberts, John S. Olson, Thomas G. Spiro

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60 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)21096-21108
Number of pages13
JournalJournal of Biological Chemistry
Issue number20
StatePublished - May 14 2004

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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