Dissociation and Recombination between Ligands and Heme in a CO-sensing Transcriptional Activator CooA

Abstract

CooA from Rhodospirillum rubrum is a transcriptional activator in which a heme prosthetic group acts as a CO sensor and regulates the activity of the protein.In this study, the electronic relaxation of the heme, and the concurrent recombination between ligands and the heme at ϳ280 K were examined in an effort to understand the environment around the heme and the dynamics of the ligands. Upon photoexcitation of the reduced CooA at 400 nm, electronic relaxation of the heme occurred with time constants of 0.8 and 1.7 ps. The ligand rebinding was substantially completed with a time constant of 6.5 ps, followed by a slow relaxation process with a time constant of 173 ps. In the case of CO-bound CooA, relaxation of the excited heme occurred with two time constants, 1.1 and 2.4 ps, which were largely similar to those with reduced CooA. The subsequent CO recombination process was remarkably fast compared with that of other CO-bound heme proteins. It was well described as a biphasic geminate recombination process with time constants of 78 ps (60%) and 386 ps (30%). About 10% of the excited heme remained unligated at 1.9 ns. The dynamics of rebinding of CO thus will help us to understand how the physiologically relevant diatomic molecule approaches the heme binding site in CooA with picosecond resolution.CooA, a transcriptional activator from Rhodospirillum rubrum, is a heme protein that acts as a CO sensor in vivo by binding . CooA is the first example of a transcriptional regulator containing heme as a prosthetic group (1). Only CObound CooA activates transcription of the genes for the key CO-oxidizing enzymes (2-6). Although the ferrous heme in CooA is in a six-coordinate form (3, 4), one of the heme axial ligands is replaced by exogenous CO upon the binding of CO (3,5,7), which triggers the conformational change in CooA required for specific binding to the target DNA (2,3,5,6,8).Though CO has been widely used as a probe to study the biochemical and biophysical properties of heme proteins, it has been thought to have no physiological role. CooA is the first example of a heme protein in which CO has a physiological function. Analysis of the dynamics of binding and escape of the ligand in heme proteins provides information on the intrinsic reactivity of the site for heme iron biding with the ligand, and how the reactivity and the pathway of the ligand are controlled by the protein. Observation of the motion of ligands such as O 2 , NO, and CO within heme proteins is facilitated by the simple photodissociation of diatom-heme protein complexes (9 -11). The dynamics of geminate rebinding, escaping, and bimolecular rebinding of ligands can be studied by various spectroscopic methods over a wide time range.Flash photolysis studies on CO-bound CooA will provide some useful information on the mechanisms of CO sensing by the heme and information on the regulation of CooA activity by CO. Measurement of transient absorption in the Soret band region on a tens of nanosecond or longer time scale has recently been carried out in studi...

Dynamics of Carbon Monoxide Binding to CooA

“…The dye laser was also used to determine the overall quantum yield for complete photolysis of CooA-CO, using MbCO as a reference (20). The value obtained, q overall Ϸ ϳ0.02 for CooA-CO, is in agreement with the more direct picosecond rebinding studies of Aono and coworkers (12)(13)(14).…”

Ligand Dynamics in an Electron Transfer Protein

“…c mutants (M80A and M80S, see Table 1). This is substantially lower than for any native ligand-binding protein, the lowest reported yields in this class of heme proteins being on the order of 5% for the CO sensor CooA from Rhodospirillum rubrum (55,56) and for the truncated hemoglobin HbO from Mycobacterium tuberculosis. 5 In addition, the fastest CO binding phases in the modified cyt.…”

Temperature-dependent heme kinetics with nonexponential binding and barrier relaxation in the absence of protein conformational substates

“…This strongly suggests that, in addition to the distal enthalpic barrier (H 0 Ϸ 7 kJ/mol), there are significant entropic barriers that are presented by the protein, which are absent in the model systems. Based on the significant variations in the Arrhenius prefactors between MbCO (1.6 ϫ 10 9 s Ϫ1 ) and FePPIX-CO (1.5 ϫ 10 11 s Ϫ1 ), and what can be deduced for other heme proteins (36,37), it appears to us that the role of entropic control in heme proteins may have been underestimated. The Arrhenius prefactor can be approximated as…”

“…An important additional question raised by these observations is whether the various models for CO binding to heme in Mb can be extended to explain the ultrafast subnanosecond geminate rebinding of CO to protoheme (3,26,35) and to various other heme proteins, such as CooA (36) or carboxymethyl cytochrome c (37), for which the Arrhenius prefactor must be ϳ10 11 s Ϫ1 (instead of 10 9 s Ϫ1 as found for MbCO). If the Arrhenius prefactors for CO binding can differ by several orders of magnitude between different heme proteins and heme environments, it suggests that entropic factors (rather than spin selection rules) may turn out to be an important and underappreciated source of biochemical control in this class of protein reactions.…”