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

Abstract

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...

Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

“…) of heme-bound PAS proteins (EcDOS and BjFixL) (40,43,46) but lower than those (0.22-1.0 M Ϫ1 s Ϫ1 ) of the globin proteins (YddV, HemAT-Bs, BpeGReg, and SWMb) (28,31,34,45). The dissociation rate constant of CO for AfGcHK was significantly lower than those (0.019 -0.067 s Ϫ1 ) of the other oxygen sensor enzymes and SWMb (45).…”

“…S4). The O 2 dissociation rate constant (0.10 s Ϫ1 ) of (43). The equilibrium dissociation constants of AfGcHK calculated from these rate constants were 0.077 and 0.67 M, which were lower than those (0.64 -14 M) of other globin proteins (28,31,34,45), suggesting that the novel histidine kinase has unusually high oxygen affinity as GCS.…”

“…The CO dissociation rate constant was estimated by mixing 5 M CO-bound AfGcHK with 0.1 mM potassium ferricyanide in 50 mM TrisHCl, pH 8.0, using a MultiSpec-1500 (Shimadzu) photodiode array spectrometer equipped with a Peltier cell temperature controller at 25°C (28,43,44).…”

“…S3) (28, 40 -42). The second-order rate constant for O 2 association with full-length AfGcHK was composed of a two phases with rate constants of 1.3 and 0.15 M Ϫ1 s Ϫ1 at a ϳ1:1 ratio ( (42,43,46) and lower than those (7-32…”

“…The pK a value of the Tyr-OH is 10.9, whereas the corresponding values for water associated with SWMb and human hemoglobin are 8.99 and 8.05, respectively (37). Based on the pH-insensitive spectral behavior of AfGcHK between pH 7.0 and 10.0, we suggest that the hydroxide is the axial ligand in the distal heme side, similar to EcDOS (20,22,43). The Fe(II) complex was a 5-coordinated high spin complex, whereas Fe(II)-O 2 -and Fe(II)-CO-bound proteins were 6-coordinated low spin complexes (Fig.…”