Marie Alda Gilles-Gonzalez

An Oxygen-Sensing Diguanylate Cyclase and Phosphodiesterase Couple for c-di-GMP Control

A commonly observed coupling of sensory domains to GGDEF-class diguanylate cyclases and EAL-class phosphodiesterases has long suggested that c-di-GMP synthesizing and degrading enzymes sense environmental signals. Nevertheless, relatively few signal ligands have been identified for these sensors, and even fewer instances of in vitro switching by ligand have been demonstrated. Here we describe an Escherichia coli two-gene operon, dosCP, for control of c-di-GMP by oxygen. In this operon, the gene encoding the oxygen-sensing c-di-GMP phosphodiesterase Ec Dos (here renamed Ec DosP) follows and is translationally coupled to a gene encoding a diguanylate cyclase, here designated DosC. We present the first characterizations of DosC and a detailed study of the ligand-dose response of DosP. Our results show that DosC is a globin-coupled sensor with an apolar but accessible heme pocket that binds oxygen with a K(d) of 20 microM. The response of DosP activation to increasing oxygen concentration is a complex function of its ligand saturation such that over 80% of the activation occurs in solutions that exceed 30% of air saturation (oxygen >75 microM). Finally, we find that DosP and DosC associate into a functional complex. We conclude that the dosCP operon encodes two oxygen sensors that cooperate in the controlled production and removal of c-di-GMP.

DosT and DevS are oxygen-switched kinases in Mycobacterium tuberculosis

Exposure of Mycobacterium tuberculosis to hypoxia is known to alter the expression of many genes, including ones thought to be involved in latency, via the transcription factor DevR (also called DosR). Two sensory kinases, DosT and DevS (also called DosS), control the activity of DevR. We show that, like DevS, DosT contains a heme cofactor within an N‐terminal GAF domain. For full‐length DosT and DevS, we determined the ligand‐binding parameters and the rates of ATP reaction with the liganded and unliganded states. In both proteins, the heme state was coupled to the kinase such that the unliganded, CO‐bound, and NO‐bound forms were active, but the O2‐bound form was inactive. Oxygen‐bound DosT was unusually inert to oxidation to the ferric state (half life in air >60 h). Though the kinase activity of DosT was unaffected by NO, this ligand bound 5000 times more avidly than O2 to DosT (Kd [NO] ∼5 nM versus Kd [O2] = 26 μM). These results demonstrate direct and specific O2 sensing by proteins in M. tuberculosis and identify for the first time a signal ligand for a sensory kinase from this organism. They also explain why exposure of M. tuberculosis to NO donors under aerobic conditions can give results identical to hypoxia, i.e., NO saturates DosT, preventing O2 binding and yielding an active kinase.

Novel Heme-based Oxygen Sensor with a Revealing Evolutionary History

To monitor fluctuations in oxygen concentration, cells use sensory proteins often containing heme cofactors. Here, we identify a new class of heme-binding oxygen sensors, reveal their unusual phylogenetic origin, and propose a sensing mode of a member of this class. We show that heme is bound noncovalently to the central region of AppA, an oxygen and light sensor from Rhodobacter sphaeroides. The addition of oxygen to ferrous AppA discoordinated the heme, and subsequent oxygen removal fully restored the heme coordination. In vitro, the extent of heme discoordination increased gradually with the rise in oxygen levels over a broad concentration range. This response correlated well with the gradual decrease in transcription of photosynthesis genes regulated by AppA and its partner repressor PpsR. We conclude that the AppA-PpsR regulatory system functions as an oxygen-dependent transcriptional rheostat. We identified a new domain embedded in the central region of AppA and designated it SCHIC for sensor containing heme instead of cobalamin. A phylogenetic analysis revealed that SCHIC domain proteins form a distinct cluster within a superfamily that includes vitamin B12-binding proteins and other proteins that may bind other kinds of tetrapyrroles.

Oxygen Signal Transduction

Although manifestations of O 2 adaptation have long been examined, only now are biochemical mechanisms of O 2 regulation beginning to be understood. This article comments on the current state of knowledge about proteins that function as direct sensors of molecular oxygen and makes predictions about as yet undiscovered sensors.

Hell's Gate globin I: An acid and thermostable bacterial hemoglobin resembling mammalian neuroglobin

Hells Gate globin I (HGbI), a heme‐containing protein structurally homologous to mammalian neuroglobins, has been identified from an acidophilic and thermophilic obligate methanotroph, Methylacidiphilum infernorum. HGbI has very high affinity for O2 and shows barely detectable autoxidation in the pH range of 5.2–8.6 and temperature range of 25–50 °C. Examination of the heme pocket by X‐ray crystallography and molecular dynamics showed that conformational movements of Tyr29(B10) and Gln50(E7), as well as structural flexibility of the GH loop and H‐helix, may play a role in modulating its ligand binding behavior. Bacterial HGbIs unique resistance to the sort of extreme acidity that would extract heme from any other hemoglobin makes it an ideal candidate for comparative structure–function studies of the expanding globin superfamily.

Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti

BACKGROUND [corrected] The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity. On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase. This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy. RESULTS Using one-dimensional NMR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively. We determined the hyperfine-shifted 1H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals. CONCLUSIONS These findings support the spin-state mechanism of FixL regulation. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.

Signal Transduction and Phosphoryl Transfer by a FixL Hybrid Kinase with Low Oxygen Affinity: Importance of the Vicinal PAS Domain and Receiver Aspartate

FixL is a prototype for heme-based sensors, multidomain proteins that typically couple a histidine protein kinase activity to a heme-binding domain for sensing of diatomic gases such as oxygen, carbon monoxide, and nitric oxide. Despite the relatively well-developed understanding of FixL, the importance of some of its domains has been unclear. To explore the impact of domain-domain interactions on oxygen sensing and signal transduction, we characterized and investigated Rhizobium etli hybrid sensor ReFixL. In ReFixL, the core heme-containing PAS domain and kinase region is preceded by an N-terminal PAS domain of unknown function and followed by a C-terminal receiver domain. The latter resembles a target substrate domain that usually occurs independently of the kinase and contains a phosphorylatable aspartate residue. We isolated the full-length ReFixL as a soluble holoprotein with a single heme b cofactor. Despite a low affinity for oxygen (K(d) for O₂ of 738 μM), the kinase activity was completely switched off by O₂ at concentrations well below the K(d). A deletion of the first PAS domain strongly increased the oxygen affinity but essentially prohibited autophosphorylation, although the truncated protein was competent to accept phosphoryl groups in trans. These studies provide new insights into histidyl-aspartyl phosphoryl transfers in two-component systems and suggest that the control of ligand affinity and signal transduction by PAS domains can be direct or indirect.

The PpaA/AerR Regulators of Photosynthesis Gene Expression from Anoxygenic Phototrophic Proteobacteria Contain Heme-Binding SCHIC Domains

The SCHIC domain of the B12-binding domain family present in the Rhodobacter sphaeroides AppA protein binds heme and senses oxygen. Here we show that the predicted SCHIC domain PpaA/AerR regulators also bind heme and respond to oxygen in vitro, despite their low sequence identity with AppA.

Oxygen‐Sensing Histidine‐Protein Kinases: Assays of Ligand Binding and Turnover of Response‐Regulator Substrates

Heme-based sensors are a recently discovered functional class of heme proteins that serve to detect physiological fluctuations in oxygen (O(2)), carbon monoxide (CO), or nitric oxide (NO). Many of these modular sensors detect heme ligands by coupling a histidine-protein kinase to a heme-binding domain. They typically bind O2, CO, and NO but respond only to one of these ligands. Usually, they are active in the ferrous unliganded state but are switched off by saturation with O2. The heme-binding domains of these kinases are quite varied. They may feature a PAS fold, as in the Bradyrhizobium japonicum and Sinorhizobium melitoti FixL proteins, or a GAF fold, as in the Mycobacterium tuberculosis DevS and DosT proteins. Alternative folds, such as HNOB (also H-NOX), have also been noted for such signal-transducing kinases, although these classes are less well studied. Histidine-protein kinases function in partnership with cognate response-regulator substrate(s): usually transcription factors that they activate by phosphorylation. For example, FixL proteins specifically phosphorylate their FixJ partners, and DevS and DosT proteins phosphorylate DevR in response to hypoxia. We present methods for purifying these sensors and their protein substrates, verifying the quality of the preparations, determining the K(d) values for binding of ligand and preparing sensors of known saturation, and measuring the rates of turnover (k(cat)) of the protein substrate by sensors of known heme status.

Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O2

Genome inspection revealed nine putative heme‐binding, FixL‐homologous proteins in Chlamydomonas reinhardtii. The heme‐binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand‐binding histidine FXL1‐H200S and FXL5‐H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O2] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand‐binding measurements showed that FXL1 and FXL5 bind O2 with moderate affinity, 135 and 222 μM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O2‐sensing mechanisms analogous to that reported in nitrogen‐fixing bacteria to regulate gene expression.