The heme environments of Met 95 and His 77 mutants of the isolated heme-bound PAS domain (Escherichia coli DOS PAS) of a direct oxygen sensing protein from E. coli (E. coli DOS) were investigated with resonance Raman (RR) spectroscopy and compared with the wild type (WT) enzyme. The RR spectra of both the reduced and oxidized WT enzyme were characteristic of six-coordinate low spin heme complexes from pH 4 to 10. The time-resolved RR spectra of the photodissociated CO-WT complex had an iron-His stretching band ( Fe-His ) at 214 cm ؊1 , and the Fe-CO versus CO plot of CO-WT E. coli DOS PAS fell on the line of His-coordinated heme proteins. The photodissociated CO-H77A mutant complex did not yield the Fe-His band but gave a Fe-Im band in the presence of imidazole. The RR spectrum of the oxidized M95A mutant was that of a six-coordinate low spin complex (i.e. the same as that of the WT enzyme), whereas the reduced mutant appeared to contain a fivecoordinate heme complex. Taken Heme-containing signal-transducing proteins (1-3) respond to diatomic molecules, which act as physiological, environmental messengers. This has attracted the attention of biophysical chemists. The O 2 sensing proteins so far identified include FixL (an oxygen-sensing kinase of Rhizobia meliloti) (1, 4), HemAT (an oxygen sensor heme protein discovered from Bacillus subtilis (HemAT-Bs) and Halobacterium salinarium (HemAT-Hs)) (5, 6), PDEA1 (7), and putatively a heme protein from E. coli (designated Escherichia coli DOS) (8). There is only one CO sensor protein known (CooA, a CO-binding transcriptional regulation factor from Rhodospirillum rubrum) (9, 10) and one NO sensor (soluble guanylate cyclase) (11,12). In each case, binding of an external ligand to the heme located in an N-terminal sensory domain transmits a signal to the functional C-terminal domain (either enzymatic or DNA binding). We are curious to know how these proteins recognize a specific diatomic molecule to generate the appropriate physiological response and what kind of structural changes occur to transmit the signal from the sensory domain to the functional domain.The sensory domain of FixL belongs to the large family of signal-transducing PAS domain 1 proteins, whereas those of HemAT, CooA, and soluble guanylate cyclase do not. The PAS domain proteins found in eukarya, archaea, and bacteria contain a partly conserved tertiary structure despite their limited sequence homology (Ͻ15%) and dissimilar cofactors (13). Although structures of three PAS proteins including the human voltage sensor (HERG) (14), the rhizobial oxygen sensor (FixL) (15, 16), and bacterial light sensor (PYP) (17) have been solved, interactions between the sensory domain and the functional domain are not clearly understood. Namely, hydrophobic interactions seem important to regulate the K ϩ channel of HERG, whereas polar interactions in the EF loop of the PAS domain seem to be essential to PYP. In the case of FixL, either a protein conformational change associated with the location of the heme iron (in-pla...

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its ␣-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be ؉67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min ؊1 with cAMP, which was optimal at pH 8.5 in the presence of Mg 2؉ and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.Heme proteins and enzymes perform a broad range of functions. Well known examples include O 2 storage with myoglobin, O 2 carriage with hemoglobin, mediators of electron transfer with cytochromes, and catalytic activation of heme ligands with P450s and peroxidases (1-3). Recently, a new class of heme enzymes involved in intramolecular signal transduction is emerging, known as heme-based sensors (4 -6). Almost of all the heme-based sensors contain two different functional domains as follows: one is an N-terminal heme domain, which acts a sensor, and the other is a catalytic domain such as a histidine kinase or a soluble guanylate cyclase. These heme sensor enzymes use the heme for mediating transcriptional and regulatory events associated with the presence of gaseous molecules such as CO, NO, and O 2 (4 -6). In these enzymes, the ligand association or dissociation from the heme iron leads to protein conformational changes, which transmit signals to the other domain where they initiate catalytic function or DNA binding. For example, the CooA 1 protein from Rhodospirillum rubrum is a CO sensor heme protein that regulates the expression of the coo genes associated with CO-dependent growth (Refs. 7 and 8 and references therein). Soluble guanylate cyclase is an NO sensor heme protein that regulates conversion of 5Ј-GTP to the intracellular second messenger, cGMP (Refs. 9 and 10 and references therein). Hem-AT-Bs and Hem-AT-Hs are oxygen sensors in which the hemes are thought to mediate signal transduction for methylation of the chemotaxis proteins (11, 12).The Fix proteins, FixL and FixJ, of Rhizobium meliloti are well characterized as biological oxygen sensors and regulate the expression of the nitrogen fixation genes of a plant symbiotic bacterium, Sino...

Biochemistry Z 0250 Structure-Function Relationships of EcDOS, a Heme-Regulated Phosphodiesterase from Escherichia coli -[65 refs.]. -(SASAKURA*, Y.; YOSHIMURA-SUZUKI, T.; KUROKAWA, H.; SHIMIZU, T.; Acc. Chem. Res. 39 (2006) 1, 37-43; Inst. Multidisc. Res. Adv. Mater., Tohoku Univ., Aoba, Sendai 980, Japan; Eng.) -Lindner 19-265

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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...

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The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144 -201) and a C-terminal catalytic domain (amino acids 336 -799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (⌬N147, amino acids 148 -807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wildtype protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg 2؉ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe 2؉ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.

Recent studies have revealed a new class of heme enzymes, the heme-based sensors, which are able to turn on or off cellular signal transduction pathways in response to environmental changes. One of these enzymes is the heme-regulated phosphodiesterase from Escherichia coli (EcDOS). This protein is composed of an N-terminal heme-containing PAS domain and a C-terminal functional domain. PAS is an acronym formed from the names of the Drosophila period clock protein (PER), vertebrate aryl hydrocarbon receptor nuclear translocator (ARNT), and Drosophila single-minded protein (SIM). The heme cofactor in its PAS domain can act as a sensor of the cellular redox state that regulates the adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase activity. The crystal structures of its heme-containing PAS domain have helped clarify how the heme redox-dependent structural changes initiate intramolecular signal transduction. Here, we review recent findings on the structure-function relationships of EcDOS.