The Heme Pocket Afforded by Gly117 Is Crucial for Proper Heme Ligation and Activity of CooA

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CooA from Rhodospirillum rubrum is a heme-containing transcriptional activator that becomes activated only upon binding CO. The basis for this specificity has been probed in a CooA variant, termed ⌬P3R4 CooA, lacking two residues adjacent to the Pro 2 heme ligand, which weakens that ligand. ⌬P3R4 CooA can bind imidazole and CN ؊ , as well as CO, and form a 6-coordinate low spin adduct with each. However, in contrast to the case with CO, imidazole and CN ؊ do not stimulate the DNA binding activity of ⌬P3R4 CooA. This result indicates that the CO-specific activation of CooA is not merely the result of creation of a 6-coordinate CooA adduct but that there must be another element to this response. One feature of CooA activation is modest repositioning of the C-helices upon CO binding, so we altered a portion of the C-helix (residues Ile 113 and Leu 116 ) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile 113 and/or Leu 116 positions are not critical for CooA activation by CO, disproving a precise interaction between these residues and the CO-bound heme as a basis for the CO activation mechanism and CO ligand specificity. In contrast, hydrophobic residues at these positions contribute to the activation. Some CooA variants altered at these positions in the background of ⌬P3R4 were also found to show low but reproducible activation in response to imidazole binding to the heme. A model for the role of hydrophobicity in CooA activation and specificity is suggested.

Section: Weakening Thementioning

confidence: 73%

Section: Methodsmentioning

confidence: 99%

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The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

Youn

Kerby

Roberts

2003

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“…Ec DOS therefore constitutes a novel class of heme enzymes designated "heme-based sensors" (3)(4)(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.…”

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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...

“…1). In the CO-bound form, the C-helix residues Ile 113 , Leu 116 , and Gly 117 have been identified to be near the bound CO by resonance Raman analysis (15), and the roles of these residues in CooA function in response to CO have been evaluated (8,16). However, the active form of CooA has not been solved structurally and therefore the molecular basis of CO specificity is unknown.…”

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confidence: 99%

Changing the Ligand Specificity of CooA, a Highly Specific Heme-based CO Sensor

Youn

Kerby

Roberts

2004

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The CO-specific heme-based sensor CooA regulates the ability of Rhodospirillum rubrum to grow on CO as an energy source. Only CO triggers the conformational change of CooA essential for the protein to function as a transcriptional activator. A structurally informed mutagenesis, followed by an in vivo screening method, allowed the isolation of a series of novel CooA variants that show very substantial response to imidazole. Compared with wild-type CooA, the ligand selectivity between imidazole and CO had been changed in some variants by roughly three orders of magnitude. Remarkably, different CooA variants also showed the ability to discriminate among imidazole derivatives, strongly implying a mechanism of precise interactions between the affected residues and the various ligands. Although wild-type CooA and imidazole-responsive CooA variants appear to recognize their respective ligands by fundamentally different mechanisms, several lines of evidence suggest that they respond by a similar C-helix repositioning that results in the rearrangement of the DNA-binding domains responsible for specific DNA contact. These results have implications for the molecular basis of both the imidazole responsiveness in the variants and the stringent CO specificity of wild-type CooA.