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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.

A highly sensitive microarray system for detecting protein-protein interactions has been developed. This method was successfully applied to analyze the interactions of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS). To immobilize (His)6-Tag fused Ec DOS, anti-(His)6-Tag monoclonal antibody (anti-(His)6-Tag mAb) was initially immobilized on the solid surface, and (His)6-Tag fused Ec DOS was fixed by antigen-antibody interactions. For this experiment, ProteoChip, generally suitable for antibody immobilization, was used as solid substrate. In this report, we confirm the antibody immobilization ability of ProteoChip and specific binding to the F(c) region of the antibody. Based on this finding, interdomain interactions between Ec DOS and the isolated heme-bound PAS domain were investigated on the solid surface. Ec DOS immobilized via anti-(His)6-Tag mAb maintained interactions with the PAS fragment, in contrast to directly immobilized Ec DOS in the absence of anti-(His)6-Tag mAb. Heme-redox-sensitive interactions between Ec DOS and the PAS fragment were additionally detected using anti-(His)6-Tag mAb as a mediator. Our results collectively suggest that the immobilization method using anti-Tag antibody is suitable for maintaining native protein characteristics to facilitate elucidation of their structures and functions on solid surfaces.

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 microM(-1) s(-1), compared with >9.5 microM(-1) s(-1) for 6-coordinated globin-type hemoproteins, as determined by the stopped-flow method. This rate is dramatically increased (up to 16-fold) in the isolated heme-bound PAS domain. Dissociation constants (K(d)) calculated from the kinetic parameters are 340 and 20 microm for the full-length wild-type enzyme and its isolated PAS domain, respectively. Mutations at Met-95 in the isolated PAS domain, which may be a heme axial ligand in the Fe(II) complex, lead to a further increase in the k(on) value by more than 30-fold, and consequently, a decrease in the K(d) value to less than 1 microM. The k(on) value for CO binding to the full-length wild-type enzyme is also very low (0.00081 microM(-1) s(-1)). The kinetics of CO binding to the isolated PAS domain and its mutants are similar to those observed for O(2). However, the K(d) values for CO are considerably lower than those for O(2).

On the basis of amino acid sequences and crystal structures of similar enzymes, it is proposed that Met95 of the heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) acts as a heme axial ligand. In accordance with this proposal, the Soret and visible optical absorption and magnetic circular dichroism spectra of the Fe(II) complexes of the Met95Ala and Met95Leu mutant proteins indicate that these complexes are five-coordinated high-spin, suggesting that Met95 is an axial ligand for the Fe(II) complex. However, the Fe(III) complexes of these mutants are six-coordinated low-spin, like the wild-type enzyme. The latter spectral findings are inconsistent with the proposal that the axial ligand to the Fe(III) heme is Met95. To determine the possibility of a redox-dependent ligand switch in Ec DOS, we further analyzed Soret CD spectra and redox potentials, which provide direct evidence on the environmental structure of the heme protein. CD spectra of Fe(III) Met95 mutants were all different from those of the wild-type protein, suggesting indirect coordination of Met95 to the Fe(III) wild-type heme. The redox potentials of the Met95Leu, Met95Ala and Met95His mutants were considerably lower than that of the wild-type enzyme (+70 mV) at -1, -26, and -122 mV vs. SHE, respectively. Thus, it is reasonable to speculate that water (or hydroxy anion) interacting with Met95, rather than Met95 itself, is the axial ligand to the Fe(III) heme.

In order to understand heme environment of a heme-regulated phosphodiesterase (Ec DOS), the binding behavior of cyanide to the Fe (III) complex was examined. Interestingly, the rate of cyanide binding to full-length Ec DOS was unusually slow with k(on)=0.0022mM(-1)s(-1), while the rate for the isolated heme domain of Ec DOS (0.045mM(-1)s(-1)) was 20-fold higher. Ala and Leu mutations at Met95, which has been suggested to be a heme axial ligand, increased the k(on) rate 11- and 8-fold, respectively, and dramatically decreased the cyanide dissociation rate from the isolated heme domain. His mutation at Met95, on the other hand, caused a 17-fold decrease in the k(on) value. We discuss the unusual cyanide binding behavior and the role of Met95 in controlling cyanide binding.

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 (nu(Fe-His)) at 214 cm(-1), and the nu(Fe-CO) versus nu(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 nu(Fe-His) band but gave a nu(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 five-coordinate heme complex. Taken together, we suggest that the heme of the reduced WT enzyme is coordinated by His(77) and Met(95), and that Met(95) is displaced by CO and O(2). Presumably, the protein conformational change that occurs upon exchange of an unknown ligand for Met(95) following heme reduction may lead to activation of the phosphodiesterase domain of E. coli DOS.

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 alpha-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.