Direct Oxygen Sensor Research Articles

1P-114 酸素センサータンパク質DOSの機能に関する軸配位子の動力学的検討(ヘム蛋白質,第46回日本生物物理学会年会)

1P-114 酸素センサータンパク質DOSの機能に関する軸配位子の動力学的検討(ヘム蛋白質,第46回日本生物物理学会年会)

Transition of Escherichia coli from Aerobic to Micro-aerobic Conditions Involves Fast and Slow Reacting Regulatory Components

Understanding life at a systems level is a major aim of biology. The bacterium Escherichia coli offers one of the best opportunities to achieve this goal. It is a metabolically versatile bacterium able to respond to changes in oxygen availability. This ability is a crucial component of its lifestyle, allowing it to thrive in aerobic external environments and under the oxygen-starved conditions of a host gut. The controlled growth conditions of chemostat culture were combined with transcript profiling to investigate transcriptome dynamics during the transition from aerobic to micro-aerobic conditions. In addition to predictable changes in transcripts encoding proteins of central metabolism, the abundances of transcripts involved in homeostasis of redox-reactive metals (Cu and Fe), and cell envelope stress were significantly altered. To gain further insight into the responses of the regulatory networks, the activities of key transcription factors during the transition to micro-aerobic conditions were inferred using a probabilistic modeling approach, which revealed that the response of the direct oxygen sensor FNR was rapid and overshot, whereas the indirect oxygen sensor ArcA reacted more slowly. Similarly, the cell envelope stress sensors RpoE and CpxR reacted rapidly and more slowly, respectively. Thus, it is suggested that combining rapid and slow reacting components in regulatory networks might be a feature of systems in which a signal is perceived by two or more functionally related transcription factors controlling overlapping regulons.

Ultraviolet Resonance Raman Evidence for Utilization of the Heme 6-Propionate Hydrogen-Bond Network in Signal Transmission from Heme to Protein in Ec DOS Protein

The direct oxygen sensor protein from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of either O2 or CO molecule to a reduced heme enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report ultraviolet resonance Raman (UVRR) spectroscopic investigations of the reduced, O2- and CO-bound forms of heme-bound PAS domain of Ec DOS. The UVRR results show that heme discriminates different ligands, resulting in altered conformations in the protein moiety. Specifically, the environment around Trp53 that contacts the 2-vinyl group of heme, is changed to a more hydrophobic environment by O2 binding, whereas it is changed to a more hydrophilic environment by CO-binding. In addition, the PDE activity of the O2- and CO-bound forms for the Trp53Phe mutant is significantly decreased compared with that of the wild type (WT), demonstrating the importance of Trp53 for the catalytic reaction. On the other hand, the binding of O2 or CO to the heme produces drastic changes in the Tyr126 of Ibeta-strand at the surface of the sensor domain. Furthermore, we found that Asn84 forms a hydrogen bond with Tyr126 either in the O2- or CO-bound forms but not in the reduced form. Finally, the PDE activities of the ligand-bound forms for Asn84Val and Tyr126Phe mutants are significantly reduced as compared with that of WT, suggesting the importance of the hydrogen-bonding network from heme 6-propionate to Tyr126 through Asn84 in signal transmission.

Pathway of Information Transmission from Heme to Protein upon Ligand Binding/Dissociation in Myoglobin Revealed by UV Resonance Raman Spectroscopy

Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O(2) to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O(2) or CO binding. On the other hand, binding of O(2) to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.

Structure of factor-inhibiting hypoxia-inducible factor 1: An asparaginyl hydroxylase involved in the hypoxic response pathway.

Precise regulation of the evolutionarily conserved hypoxia-inducible transcription factor (HIF) ensures proper adaptation to variations in oxygen availability throughout development and into adulthood. Oxygen-dependent regulation of HIF stability and activity are mediated by hydroxylation of conserved proline and asparagine residues, respectively. Because the relevant prolyl and asparginyl hydroxylases use O(2) to effect these posttranslational modifications, these enzymes are implicated as direct oxygen sensors in the mammalian hypoxic response pathway. Here we present the structure of factor-inhibiting HIF-1 (FIH-1), the pertinent asparaginyl hydroxylase involved in hypoxic signaling. Hydroxylation of the C-terminal transactivation domain (CTAD) of HIF by FIH-1 prevents CTAD association with transcriptional coactivators under normoxic conditions. Consistent with other structurally known hydroxylases, FIH-1 is comprised of a beta-strand jellyroll core with both Fe(II) and the cosubstrate 2-oxoglutarate bound in the active site. Details of the molecular contacts at the active site of FIH-1 have been elucidated and provide a platform for future drug design. Furthermore, the structure reveals the presence of a FIH-1 homodimer that forms in solution and is essential for FIH activity.

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.

Characterization of a Direct Oxygen Sensor Heme Protein fromEscherichia coli: EFFECTS OF THE HEME REDOX STATES AND MUTATIONS AT THE HEME-BINDING SITE ON CATALYSIS AND STRUCTURE

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.

Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor.

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.

FNR is a direct oxygen sensor having a biphasic response curve

FNR is a transcription regulator that controls the expression of target genes in response to anoxia. Anaerobiosis is accompanied by the acquisition of two [4Fe-4S] 2+ clusters per FNR dimer and the ability to bind DNA site-specifically. Oxidation of the [4Fe-4S] 2+ form of FNR by O 2 produced a non-DNA-binding, transcriptionally inactive form which also contains an iron-sulfur cluster, recently identified by Mossbauer spectroscopy as a [2Fe-2S] cluster (Khoroshilova et al., 1997, PNAS. 94, 6078). Complete conversion needed at least 2.5–3.0 molecules of O 2 per [4Fe-4S] 2+ cluster. Using sub-stoicheiometric amounts of air-saturated buffer, stable equilibria were established in which the [4Fe-4S] 2+ and [2Fe-2S] 2+ forms co-exist and no EPR detectable free ferric ions were released. In contrast, a 20-fold molar excess K 3Fe(CN) 6 was required to oxidise the [4Fe-4S] 2+ cluster and in this case, ferric ions were released. FNR is therefore a sensitive O 2 sensor.