Heme Environmental Structure of CooA Is Modulated by the Target DNA Binding

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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...

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confidence: 77%

Section: Distinction Between Relaxation Of Heme and Ligandmentioning

confidence: 99%

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confidence: 99%

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

Kumazaki

Nakajima

Sakaguchi

et al. 2000

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“…CO may interact with a distal His in myoglobin and hemoglobin, but does not bind similarly to distal amino acids in CooA (48) and P. caudatum Hb (49). Therefore, CO is unlikely to interact directly with distal amino acids in Ec DOS.…”

Section: Fig 6 Ft Ir Spectra Of Co Complexes Of the Wild Type (--)mentioning

confidence: 99%

Binding of Oxygen and Carbon Monoxide to a Heme-regulated Phosphodiesterase from Escherichia coli

Taguchi

Matsui

Igarashi

et al. 2004

The heme-regulated phosphodiesterase, Ec DOS, is a redox sensor that uses the heme in its PAS domain to regulate catalysis. The rate of O 2 association (k on ) with full-length Ec DOS is extremely slow at 0. 0019 The phosphodiesterase (PDE) 1 from Escherichia coli, Ec DOS, is composed of an N-terminal heme-bound PAS domain and a C-terminal PDE catalytic domain (1). The basic physicochemical characteristics and function of this enzyme have been partially elucidated by our group and that of Kitagawa et al. (1,2). PDE activity is dependent on the redox state of Ec DOS in that the enzyme is active only when the heme is in the Fe(II) state. Changes in the redox state of the heme bound to the N-terminal PAS domain may induce a subtle conformational change, which intramolecularly transmits signals to the C-terminal PDE domain to initiate and/or regulate catalysis. Ec DOS therefore constitutes a novel class of heme enzymes designated "heme-based sensors" (3-5). These include proteins such as FixL (6, 7), CooA (8, 9), sGC (10, 11), and Hem-AT (12, 13). In these enzymes, association or dissociation of the exogenous axial ligand (O 2 , CO, or NO) from the heme iron leads to protein conformational changes, which in turn transmit signals to other domains to regulate catalysis or binding to DNA. The Ec DOS signal transducing mechanism appears to be unique, since changes in the redox state of the PAS domain, rather than iron coordination chemistry, are responsible for signal transduction (1). However, in other words, signal transduction triggered by ligand binding (CO and NO) is common to Ec DOS and FixL, based on the finding that CO or NO binding abolishes catalysis by Ec DOS (1).The physicochemical properties of the isolated heme-bound PAS domain of Ec DOS were initially characterized by GillesGonzales and colleagues (14). The kinetics of exogenous axial ligand binding to the heme protein and associated equilibrium constants provide useful information on the structure and characteristics of the heme distal site and ligand access channel (16 -19). It is important to study O 2 and CO binding, particularly to full-length Ec DOS, to clarify whether the enzyme is a direct O 2 sensor. These analyses would also be useful in elucidating the structure of the heme distal site and ligand access channel and their relation to the signal transduction mechanism. Based on the amino acid sequence alignment and crystal structure of a similar PAS enzyme, FixL (7,20,21), Met-95 is suggested as a heme axial ligand trans to His-77.In the present study, we report rate and equilibrium constants for O 2 and CO binding to the full-length enzyme in the Fe(II) state, isolated heme-bound PAS domain, and Met-95 * 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.‡ To whom correspondence should be addressed: Institute of Multidisciplinary Research for Advanced Materials, Tohok...

“…Replacement of one of the axial ligands of the ferrous heme with CO triggers the activation of CooA (28, 29, 34 -36). The release of the axial ligand from the heme upon binding CO causes conformational changes around the heme and subsequently in the whole molecule (28,29,35). These conformational changes induced by the interchange of the axial ligand are the principal part of the activation of CooA by CO.…”

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Redox Properties and Coordination Structure of the Heme in the CO-sensing Transcriptional Activator CooA

Nakajima

Honma

Tawara

et al. 2001

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