Atsunari Tanaka

Important roles of Tyr43 at the putative heme distal side in the oxygen recognition and stability of the Fe(II)-O2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase.

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.

Critical Role of the Heme Axial Ligand, Met95, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O2 or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg97 in the heme distal side in Ec DOS interacts with the O2 molecule and Met95 serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O2 complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O2. In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O2, CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met95 coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met95 caused by the binding of the external ligands or mutations at Met95 releases the catalytic lock and activates catalysis.

Arg97 at the Heme-Distal Side of the Isolated Heme-Bound PAS Domain of a Heme-Based Oxygen Sensor from Escherichia coli (Ec DOS) Plays Critical Roles in Autoxidation and Binding to Gases, Particularly O2

The catalytic activity of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) on cyclic di-GMP is markedly enhanced upon binding of gas molecules, such as O2 and CO, to the heme iron complex in the sensor domain. Arg97 interacts directly with O2 bound to Fe(II) heme in the crystal structure of the isolated heme-bound sensor domain with the PAS structure (Ec DOS-PAS) and may thus be critical in ligand recognition. To establish the specific role of Arg97, we generated Arg97Ala, Arg97Glu, and Arg97Ile mutant Ec DOS-PAS proteins and examined binding to O2, CO, and cyanide, as well as redox potentials. The autoxidation rates of the Arg97Ala and Arg97Glu mutant proteins were up to 2000-fold higher, while the O2 dissociation rate constant for dissociation from the Fe(II)-O2 heme complex of the Arg97Ile mutant was 100-fold higher than that of the wild-type protein. In contrast, the redox potential values of the mutant proteins were only slightly different from that of the wild type (within 10 mV). Accordingly, we propose that Arg97 plays critical roles in recognition of the O2 molecule and redox switching by stabilizing the Fe(II)-O2 complex, thereby anchoring O2 to the heme iron and lowering the autoxidation rate to prevent formation of Fe(III) hemin species not regulated by gas molecules. Arg97 mutations significantly influenced interactions with the internal ligand Met95, during CO binding to the Fe(II) complex. Moreover, the binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins was similar to that of O2, which is evident from the Kd values, suggestive of electrostatic interactions between cyanide and Arg97.

O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form

FixL, a rhizobial heme-based O2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O2 tension, while the kinase activity is inhibited in the case of the O2-bound form. The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported. Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations. The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage. On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O2 from CO and NO, and signals the O2-bound state by downregulating the phosphoryl transfer reaction.

Ligand binding to the Fe(III)-protoporphyrin IX complex of phosphodiesterase from Escherichia coli (Ec DOS) markedly enhances catalysis of cyclic di-GMP: roles of Met95, Arg97, and Phe113 of the putative heme distal side in catalytic regulation and ligand binding.

Phosphodiesterase (Ec DOS) from Escherichia coli is a gas-sensor enzyme in which binding of gas molecules, such as O(2), CO, and NO, to the Fe(II)-protoporphyrin IX complex in the sensor domain stimulates phosphodiesterase activity toward cyclic-di-GMP. In this study, we report that external axial ligands, such as cyanide or imidazole, bind to Fe(III)-protoporphyrin IX in the sensor domain and induce a 10- to 11-fold increase (from 8.1 up to 86 min(-1)) in catalysis, which is more substantial than that (6.3 to 7.2-fold) observed for other gas-stimulated Fe(II) heme-bound enzymes. Catalytic activity (50 min(-1)) of the heme-free mutant, H77A, was comparable to that of the ligand-stimulated enzymes. Accordingly, we propose that the heme at the sensor domain inhibits catalysis and that ligand binding to the heme iron complex releases this catalytic suppression. Furthermore, mutations of Met95, Arg97, and Phe113 at the putative heme distal side suppressed the ligand effects on catalysis. The rate constants (19,000 x 10(-5) microM(-1)min(-1)) for cyanide binding to the M95A and M95L mutants of the full-length enzyme were 633-fold higher than that to wild-type Ec DOS (30 x 10(-5) microM(-1)min(-1)). The absorption spectrum of the F113Y mutant suggests that the Tyr O(-) group directly coordinates to the Fe(III) complex and that the cyanide binding rate to the mutant is very slow, compared with those of the wild-type and other mutant proteins. We observed a similar trend in the binding behavior of imidazole to full-length mutant enzymes. Therefore, while Met95 and Phe113 are not direct axial ligands for the Fe(III) complex, catalytic, spectroscopic, and ligand binding evidence suggests that these residues are located in the vicinity of the heme.

Catalysis and oxygen binding of Ec DOS: a haem-based oxygen-sensor enzyme from Escherichia coli

A phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a novel haem-based oxygen sensor enzyme. Binding of O(2) to the reduced haem in the sensor domain enhances PDE activity exerted by the catalytic domain. Kinetic analysis of oxygen-dependent catalytic enhancement showed a sigmoidal curve with a Hill coefficient value of 2.8. To establish the molecular mechanism underlying allosteric regulation, we analysed binding of the O(2) ligand following reduction of haem in the isolated dimeric sensor domain using pulse radiolysis. Spectral changes accompanying O(2) binding were composed of two phases as a result of reduction of two haem complexes when high-dose electron beams were applied. In contrast, upon reduction of the dimer with a low-dose beam, the kinetics of O(2) ligation displayed single-phase behaviour as a result of the reduction of one haem complex within dimer. Based on these results, we propose that the faster phase corresponds to binding of the first O(2) molecule to one subunit of the dimer, followed by binding of the second O(2) molecule to the other subunit. Notably, for the haem axial ligand mutant proteins, M95A and M95L, O(2) binding displayed single-phase kinetics and was independent of electron beam dose.

Roles of Arg-97 and Phe-113 in regulation of distal ligand binding to heme in the sensor domain of Ec DOS protein. Resonance Raman and mutation study.

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O2, CO, or NO to a reduced heme significantly enhances the PDE activity toward 3′,5′-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 → Ile, Met-95 → Ala, Arg-97 → Ile, Arg-97 → Ala, Arg-97 → Glu, Phe-113 → Leu, and Phe-113 → Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.

Hydrogen sulfide stimulates the catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS)

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is an oxygen sensor enzyme composed of a heme-bound O(2) sensor domain at the N-terminus and a catalytic domain at the C-terminus. The catalytic activity of Ec DOS is substantially enhanced with the formation of a Fe(II) heme-O(2) complex. The physiological importance of H(2)S as a fourth signaling gas molecule in addition to O(2), CO and NO is an emerging focus of research, since H(2)S participates in various physiological functions. In the present study, we showed that catalysis by Ec DOS is markedly increased by H(2)S under aerobic conditions. Absorption spectral findings suggest that SH(-)-modified heme iron complexes, such as Fe(III)-SH(-) and Fe(II)-O(2) complexes, represent the active species for H(2)S-induced catalysis. We further examined the role of Cys residues in H(2)S-induced catalysis using Cys→Ala mutant enzymes. Based on the collective data, we speculate that H(2)S-induced catalytic enhancement is facilitated by an admixture of Fe(III)-SH(-) and Fe(II)-O(2) complexes formed during catalysis and modification of specific Cys residue(s) in the catalytic domain.

Role of Phe113 at the distal side of the heme domain of an oxygen-sensor (Ec DOS) in the characterization of the heme environment

The heme-based oxygen-sensor enzyme from Escherichia coli (Ec DOS) is a heme-regulated phosphodiesterase with activity on cyclic-di-GMP and is composed of an N-terminal heme-bound sensor domain with the PAS structure and a C-terminal functional domain. The activity of Ec DOS is substantially enhanced by the binding of O(2) to the Fe(II)-protoporphyrin IX complex [Fe(II) complex] in the sensor domain. The binding of O(2) to the Fe(II) complex changes the structure of the sensor domain, and this altered structure becomes a signal that is transduced to the functional domain to trigger catalysis. The first step in intra-molecular signal transduction is the binding of O(2) to the Fe(II) complex, and detailed elucidation of this molecular mechanism is thus worthy of exploration. The X-ray crystal structure reveals that Phe113 is located close to the O(2) molecule bound to the Fe(II) complex in the sensor domain. Here, we found that the O(2) association rate constants (>200x10(-3) microM(-1)s(-1): F113L; 26x10(-3) microM(-1)s(-1): F113Y) of the Fe(II) complexes of Phe113 mutants were markedly different from that (51x10(-3) microM(-1)s(-1)) of the wild-type enzyme, and auto-oxidation rates (0.00068 min(-1): F113L; 0.039 min(-1): F113Y) of the Phe113 mutants also differed greatly from that (0.0062 min(-1)) of the wild-type enzyme. We thus suggest that Phe113, residing near the O(2) molecule, has a critical role in optimizing the Fe(II)-O(2) complex for effective regulation of catalysis by the oxygen-sensor enzyme. Interactions of CO and cyanide anion with the mutant proteins were also studied.