Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli

In the heme-based oxygen sensor Dos from Escherichia coli, one of the axial ligands (Met 95) of a six-coordinate heme can be replaced by external ligands such as O(2), NO, and CO, which causes a switch in phosphodiesterase activity. To gain insight into the bidirectional switching mechanism, we have studied the interaction of ligands with the sensor domain DosH by flash photolysis experiments with femtosecond time resolution. The internal ligand can be photodissociated from the ferrous heme and recombines with time constants of 7 and 35 ps. This is somewhat slower than recombination of the external ligands NO, with which picosecond rebinding occurs with unprecedented efficiency (>99%) with a predominant phase of approximately 5 ps, and O(2) (97% in 5 ps, Liebl, U., Bouzhir-Sima, L., Négrerie, M., Martin, J.-L., and Vos, M. H. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 12771-12776). Dissociated CO displays geminate rebinding in 1.5 ns with a very high yield (60%). Together these results indicate that the heme environment provides a very tight pocket for external ligands, presumably preventing frequent switching events. Additional CO dissociation and rebinding experiments on a longer time scale reveal that (a) Met 95 binding, in 100 micros, occurs in competition with bimolecular CO binding, and (b) subsequent replacement of Met 95 by CO on the millisecond time scale occurs faster than in rapid-mixing experiments, suggesting a slow further relaxation. A minimal ligand binding model is proposed that suggests that Met 95 displacement from the heme is facilitated by the presence of an external ligand in the heme environment. Furthermore, the orders of magnitude difference between Met 95 binding after dissociation of internal and external ligands, as well as the spectral characteristics of photodissociation intermediates, indicate substantial rearrangement of the heme environment associated with ligand sensing. Further remarkable observations include evidence for stable (>4 ns) photooxidation of six-coordinate ferrous heme, with a quantum yield of 4-8%.     

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 O(2), 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.     

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.     

pH dependence of cyanide binding to the ferric heme domain of the direct oxygen sensor from Escherichia coli and the effect of alkaline denaturation

The spectrum of the ferric heme domain of the direct oxygen sensor protein from Escherichia coli ( EcDosH) has been measured between pH 3.0 and 12.6. EcDosH undergoes acid denaturation with an apparent p K a of 4.24 +/- 0.05 and a Hill coefficient of 3.1 +/- 0.6 and reversible alkaline denaturation with a p K a of 9.86 +/- 0.04 and a Hill coefficient of 1.1 +/- 0.1. Cyanide binding to EcDosH has been investigated between pH 4 and 11. The EcDosH-cyanide complex is most stable at pH 9 with a K D of 0.29 +/- 0.06 microM. The kinetics of cyanide binding are monophasic between pH 4 and 8. At pH >or=8.5, the reaction is biphasic with the fast phase dependent upon the cyanide concentration and the slow phase independent of cyanide. The slow phase is attributed to conversion of denatured EcDosH to the native state, with a pH-independent rate of 0.052 +/- 0.006 s (-1). The apparent association rate constant for cyanide binding to EcDosH increases from 3.6 +/- 0.1 M (-1) s (-1) at pH 4 to 520 +/- 20 M (-1) s (-1) at pH 11. The dissociation rate constant averages (8.6 +/- 1.3) x 10 (-5) s (-1) between pH 5 and 9, increasing to (1.4 +/- 0.1) x 10 (-3) s (-1) at pH 4 and (2.5 +/- 0.1) x 10 (-3) s (-1) at pH 12.2. The mechanism of cyanide binding is consistent with preferential binding of the cyanide anion to native EcDosH. The reactions of imidazole and H 2O 2 with ferric EcDosH were also investigated and show little reactivity.     

Stationary and time-resolved resonance Raman spectra of His77 and Met95 mutants of the isolated heme domain of a direct oxygen sensor from Escherichia coli

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.     

Binding of oxygen and carbon monoxide to a heme-regulated phosphodiesterase from Escherichia coli. Kinetics and infrared spectra of the full-length wild-type enzyme, isolated PAS domain, and Met-95 mutants

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).     

New roles for key residues in helices H1 and H2 of the Escherichia coli H-NS N-terminal domain: H-NS dimer stabilization and Hha binding

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.