RESUMEN
In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica, an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d, a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d, and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor.
Asunto(s)
Hemo , Shewanella , Shewanella/metabolismo , Shewanella/química , Hemo/química , Hemo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Cristalografía por Rayos X , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/metabolismoRESUMEN
The globin superfamily of proteins is ancient and diverse. Regular assessments based on the increasing number of available genome sequences have elaborated on a complex evolutionary history. In this review, we present a summary of a decade of advances in characterising the globins of cyanobacteria and green algae. The focus is on haem-containing globins with an emphasis on recent experimental developments, which reinforce links to nitrogen metabolism and nitrosative stress response in addition to dioxygen management. Mention is made of globins that do not bind haem to provide an encompassing view of the superfamily and perspective on the field. It is reiterated that an effort toward phenotypical and in-vivo characterisation is needed to elucidate the many roles that these versatile proteins fulfil in oxygenic photosynthetic microbes. It is also proposed that globins from oxygenic organisms are promising proteins for applications in the biotechnology arena.
Asunto(s)
Chlorophyta , Cianobacterias , Globinas , Cianobacterias/metabolismo , Cianobacterias/genética , Chlorophyta/metabolismo , Chlorophyta/genética , Globinas/genética , Globinas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Hemo/metabolismo , Nitrógeno/metabolismo , FotosíntesisRESUMEN
Artificial proteins representing the consensus of a set of homologous sequences have attracted attention for their increased thermodynamic stability and conserved activity. Here, we applied the consensus approach to a b-type heme-binding protein to inspect the contribution of a dissociable cofactor to enhanced stability and the chemical consequences of creating a generic heme environment. We targeted the group 1 truncated hemoglobin (TrHb1) subfamily of proteins for their small size (â¼120 residues) and ease of characterization. The primary structure, derived from a curated set of â¼300 representative sequences, yielded a highly soluble consensus globin (cGlbN) enriched in acidic residues. Optical and NMR spectroscopies revealed high-affinity heme binding in the expected site and in two orientations. At neutral pH, proximal and distal iron coordination was achieved with a pair of histidine residues, as observed in some natural TrHb1s, and with labile ligation on the distal side. As opposed to studied TrHb1s, which undergo additional folding upon heme binding, cGlbN displayed the same extent of secondary structure whether the heme was associated with the protein or not. Denaturation required guanidine hydrochloride and showed that apo- and holoprotein unfolded in two transitions-the first (occurring with a midpoint of â¼2 M) was shifted to higher denaturant concentration in the holoprotein (â¼3.7 M) and reflected stabilization due to heme binding, while the second transition (â¼6.2 M) was common to both forms. Thus, the consensus sequence stabilized the protein but exposed the existence of two separately cooperative subdomains within the globin architecture, masked as one single domain in TrHb1s with typical stabilities. The results suggested ways in which specific chemical or thermodynamic features may be controlled in artificial heme proteins.
Asunto(s)
Globinas , Hemoproteínas , Globinas/química , Pliegue de Proteína , Termodinámica , Hemo/metabolismo , Desnaturalización ProteicaRESUMEN
THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.
Asunto(s)
Proteínas Algáceas/metabolismo , Lisina/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/genética , Chlamydomonas reinhardtii/química , Teoría Funcional de la Densidad , Hierro/química , Hierro/metabolismo , Lisina/química , Modelos Químicos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Conformación Proteica , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMEN
THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate-protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions.
Asunto(s)
Hemo/química , Hierro/química , Lisina/química , Hemoglobinas Truncadas/química , Chlamydomonas reinhardtii , Cristalografía por Rayos X/métodos , Compuestos Férricos/química , Hemoglobinas/química , Histidina/química , Concentración de Iones de Hidrógeno , Ligandos , Espectroscopía de Resonancia Magnética/métodos , Oxidación-Reducción , Oxigenasas/metabolismo , Conformación ProteicaRESUMEN
Amino acid replacement is a useful strategy to assess the roles of axial heme ligands in the function of native heme proteins. THB1, the protein product of the Chlamydomonas reinhardtii THB1 gene, is a group 1 truncated hemoglobin that uses a lysine residue in the E helix (Lys53, at position E10 by reference to myoglobin) as an iron ligand at neutral pH. Phylogenetic evidence shows that many homologous proteins have a histidine, methionine or arginine at the same position. In THB1, these amino acids would each be expected to convey distinct reactive properties if replacing the native lysine as an axial ligand. To explore the ability of the group 1 truncated Hb fold to support alternative ligation schemes and distal pocket conformations, the properties of the THB1 variants K53A as a control, K53H, K53M, and K53R were investigated by electronic absorption, EPR, and NMR spectroscopies. We found that His53 is capable of heme ligation in both the Fe(III) and Fe(II) states, that Met53 can coordinate only in the Fe(II) state, and that Arg53 stabilizes a hydroxide ligand in the Fe(III) state. The data illustrate that the group 1 truncated Hb fold can tolerate diverse rearrangement of the heme environment and has a strong tendency to use two protein side chains as iron ligands despite accompanying structural perturbations. Access to various redox pairs and different responses to pH make this protein an excellent test case for energetic and dynamic studies of heme ligation.
Asunto(s)
Sustitución de Aminoácidos , Hemo/química , Hemoglobinas/química , Lisina/química , Simulación de Dinámica Molecular , Proteínas de Plantas/química , Chlamydomonas reinhardtii , Hemo/genética , Lisina/genética , Conformación Proteica , Estabilidad ProteicaRESUMEN
Heme ligation in hemoglobin is typically assumed by the "proximal" histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a "distal" histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme-protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Hemoglobinas/química , Synechocystis/química , Hemoglobinas Truncadas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Hemo/genética , Hemo/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Hemoglobinas Truncadas/genética , Hemoglobinas Truncadas/metabolismoRESUMEN
BACKGROUND: The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1-4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins. METHODS: We inspected properties of the globin domains prepared by recombinant means through site-directed mutagenesis, electronic absorption, CD, and NMR spectroscopies, and X-ray crystallography. RESULTS: Recombinant THB3, which lacks the proximal histidine but has a distal histidine, binds heme weakly. NMR data demonstrate that the recombinant domains of THB2 and THB4 coordinate the ferrous heme iron with the proximal histidine and a lysine from the distal helix. An X-ray structure of ferric THB4 confirms lysine coordination. THB1, THB2, and THB4 have reduction potentials between -65 and -100 mV, are capable of nitric oxide dioxygenation, are reduced at different rates by the diaphorase domain of C. reinhardtii nitrate reductase, and show different response to peroxide treatment. CONCLUSIONS: Three single-domain C. reinhardtii hemoglobins use lysine as a distal heme ligand in both Fe(III) and Fe(II) oxidation states. This common feature is likely related to enzymatic activity in the management of reactive oxygen species. GENERAL SIGNIFICANCE: Primary structure analysis of hemoglobins has limited power in the prediction of heme ligation. Experimental determination reveals variations in this essential property across the superfamily.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Hemo/metabolismo , Lisina/metabolismo , Hemoglobinas Truncadas/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Cristalografía por Rayos X , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Ligandos , Mutagénesis Sitio-Dirigida , Óxido Nítrico/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Conformación Proteica , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMEN
The hemoglobin of Synechococcus sp. PCC 7002, GlbN, is a monomeric group I truncated protein (TrHb1) that coordinates the heme iron with two histidine ligands at neutral pH. One of these is the distal histidine (His46), a residue that can be displaced by dioxygen and other small molecules. Here, we show with mutagenesis, electronic absorption spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy that at high pH and exclusively in the ferrous state, Lys42 competes with His46 for the iron coordination site. When b heme is originally present, the population of the lysine-bound species remains too small for detailed characterization; however, the population can be increased significantly by using dimethyl-esterified heme. Electronic absorption and NMR spectroscopies showed that the reversible ligand switching process occurs with an apparent pKa of 9.3 and a Lys-ligated population of â¼60% at the basic pH limit in the modified holoprotein. The switching rate, which is slow on the chemical shift time scale, was estimated to be 20-30 s-1 by NMR exchange spectroscopy. Lys42-His46 competition and attendant conformational rearrangement appeared to be related to weakened bis-histidine ligation and enhanced backbone dynamics in the ferrous protein. The pH- and redox-dependent ligand exchange process observed in GlbN illustrates the structural plasticity allowed by the TrHb1 fold and demonstrates the importance of electrostatic interactions at the heme periphery for achieving axial ligand selection. An analogy is drawn to the alkaline transition of cytochrome c, in which Lys-Met competition is detected at alkaline pH, but, in contrast to GlbN, in the ferric state only.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Synechococcus/química , Hemoglobinas Truncadas/química , Secuencia de Aminoácidos , Complejos de Coordinación/química , Esterificación , Histidina/química , Concentración de Iones de Hidrógeno , Hierro/química , Lisina/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Presión , Propionatos , Conformación Proteica , Pliegue de Proteína , Protoporfirinas/química , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme-histidine C-N linkage similar to the C-S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII-NO) that resist reductive nitrosylation to the paramagnetic FeII-NO forms. Dithionite reduction of FeIII-NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII-NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII-H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine-heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Óxido Nítrico/metabolismo , Hemoglobinas Truncadas/química , Ligandos , Espectroscopía de Resonancia Magnética , Óxidos de Nitrógeno/metabolismo , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Oxígeno/química , Oxigenasas/química , Oxigenasas/metabolismo , Procesamiento Proteico-Postraduccional , Synechococcus/química , Synechocystis/químicaRESUMEN
Nitrate metabolism in Chlamydomonas reinhardtii involves THB1, a monomeric hemoglobin thought to function as a nitric oxide dioxygenase (NOD). NOD activity requires dioxygen and nitric oxide binding followed by a one-electron oxidation of the heme iron and nitrate release. Unlike pentacoordinate flavohemoglobins, which are efficient NODs, THB1 uses two iron axial ligands: the conserved proximal histidine and a distal lysine (Lys53). As a ligand in both the oxidized (ferric) and reduced (ferrous) states, Lys53 is expected to lower the reorganization energy associated with electron transfer and therefore facilitate reduction of the ferric enzyme. In ferrous THB1, however, Lys53 must be displaced for substrate binding. To characterize Lys53 dynamics, THB1 was studied at various pH, temperatures, and pressures by NMR spectroscopy. Structural information indicates that the protein fold and Lys53 environment are independent of the oxidation state. High-pressure NMR experiments provided evidence that displacement of Lys53 occurs through fast equilibrium (â¼3-4 × 103 s-1 at 1 bar, 298 K) with a low-population intermediate in which Lys53 is neutral and decoordinated. Once decoordinated, Lys53 is able to orient toward solvent and become protonated. The global lysine decoordination/reorientation/protonation processes measured by 15Nz-exchange spectroscopy are slow on the chemical shift time scale (101-102 s-1 at pH ≈ 6.5, 298 K) in both iron redox states. Thus, reorientation/protonation steps in ferrous THB1 appear to present a significant barrier for dioxygen binding, and consequently, NOD turnover. The results illustrate the role of distal ligand dynamics in regulating the kinetics of multistep heme redox reactions.
Asunto(s)
Proteínas Algáceas/química , Chlamydomonas reinhardtii/química , Hemo/química , Hemoglobinas/química , Lisina/química , Oxigenasas/química , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Clonación Molecular , Transporte de Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hemo/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Histidina/química , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Lisina/metabolismo , Modelos Moleculares , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , TermodinámicaRESUMEN
In the last few years, advances in algal research have identified the participation of haemoglobins in nitrogen metabolism and the management of reactive nitrogen and oxygen species. This chapter summarises the state of knowledge concerning algal haemoglobins with a focus on the most widely used model system, namely, Chlamydomonas reinhardtii. Genetic, physiologic, structural, and chemical information is compiled to provide a framework for further studies.
Asunto(s)
Proteínas Algáceas/química , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/enzimología , Hemoglobinas/química , Hemoglobinas/metabolismo , Proteínas Algáceas/genética , Hemoglobinas/genética , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
In addition to its well-known roles as an electrophile and general acid, the side chain of histidine often serves as a hydrogen bond (H-bond) acceptor. These H-bonds provide a convenient pH-dependent switch for local structure and functional motifs. In hundreds of instances, a histidine caps the N-terminus of α- and 310-helices by forming a backbone NH···Nδ1 H-bond. To characterize the resilience and dynamics of the histidine cap, we measured the trans H-bond scalar coupling constant, (2h)JNN, in several forms of Group 1 truncated hemoglobins and cytochrome b5. The set of 19 measured (2h)JNN values were between 4.0 and 5.4 Hz, generally smaller than in nucleic acids (~6-10 Hz) and indicative of longer, weaker bonds in the studied proteins. A positive linear correlation between (2h)JNN and the difference in imidazole ring (15)N chemical shift (Δ(15)N = |δ(15)Nδ1 - δ(15)Nε2|) was found to be consistent with variable H-bond length and variable cap population related to the ionization of histidine in the capping and noncapping states. The relative ease of (2h)JNN detection suggests that this parameter can become part of the standard arsenal for describing histidines in helix caps and other key structural and catalytic elements involving NH···N H-bonds. The combined nucleic acid and protein data extend the utility of (2h)JNN as a sensitive marker of local structural, dynamic, and thermodynamic properties in biomolecules.
Asunto(s)
Histidina/química , Proteínas/química , Hemoglobinas Truncadas/química , Proteínas Bacterianas/química , Chlamydomonas/química , Citocromos b5/química , Hemo/química , Hemoglobinas/química , Enlace de Hidrógeno , Metahemoglobina/análogos & derivados , Metahemoglobina/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Synechococcus/química , Synechocystis/químicaRESUMEN
THB1 is one of several group 1 truncated hemoglobins (TrHb1s) encoded in the genome of the unicellular green alga Chlamydomonas reinhardtii. THB1 expression is under the control of NIT2, the master regulator of nitrate assimilation, which also controls the expression of the only nitrate reductase in the cell, NIT1. In vitro and physiological evidence suggests that THB1 converts the nitric oxide generated by NIT1 into nitrate. To aid in the elucidation of the function and mechanism of THB1, the structure of the protein was solved in the ferric state. THB1 resembles other TrHb1s, but also exhibits distinct features associated with the coordination of the heme iron by a histidine (proximal) and a lysine (distal). The new structure illustrates the versatility of the TrHb1 fold, suggests factors that stabilize the axial ligation of a lysine, and highlights the difficulty of predicting the identity of the distal ligand, if any, in this group of proteins.
Asunto(s)
Proteínas Algáceas/química , Chlamydomonas reinhardtii/química , Hemo/química , Histidina/química , Lisina/química , Nitrato-Reductasa/química , Hemoglobinas Truncadas/química , Proteínas Algáceas/genética , Secuencias de Aminoácidos , Chlamydomonas reinhardtii/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Hierro/química , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nitrato-Reductasa/genética , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología Estructural de Proteína , Hemoglobinas Truncadas/genéticaRESUMEN
In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the Nδ1 atom of histidine and both the backbone N-H and side-chain Oγ-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of (2h)J(NN) HBCs supports that Thr N-H···Nδ1 His H-bonds within internal repeats are stronger (â¼4 Hz) than in the solvent exposed C-terminal AR (â¼2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant (2h)J and (1h)J couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in (2h)J(NN), attributed to optimization of the Val N-H···Nδ1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.
Asunto(s)
Repetición de Anquirina , Ancirinas/química , Resonancia Magnética Nuclear Biomolecular , Enlace de Hidrógeno , Modelos Moleculares , Teoría CuánticaRESUMEN
The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine-heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (>90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His-heme modification will be applicable to the design of proteins with a non-dissociable heme group.
Asunto(s)
Proteínas Bacterianas/química , Hemo/química , Histidina/química , Proteínas de Plantas/química , Ingeniería de Proteínas , Protoporfirinas/química , Hemoglobinas Truncadas/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Chlamydomonas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Synechococcus/química , Synechocystis/química , Hemoglobinas Truncadas/genéticaRESUMEN
The nuclear genome of the model organism Chlamydomonas reinhardtii contains genes for a dozen hemoglobins of the truncated lineage. Of those, THB1 is known to be expressed, but the product and its function have not yet been characterized. We present mutagenesis, optical, and nuclear magnetic resonance data for the recombinant protein and show that at pH near neutral in the absence of added ligand, THB1 coordinates the heme iron with the canonical proximal histidine and a distal lysine. In the cyanomet state, THB1 is structurally similar to other known truncated hemoglobins, particularly the heme domain of Chlamydomonas eugametos LI637, a light-induced chloroplastic hemoglobin. Recombinant THB1 is capable of binding nitric oxide (NO(â¢)) in either the ferric or ferrous state and has efficient NO(â¢) dioxygenase activity. By using different C. reinhardtii strains and growth conditions, we demonstrate that the expression of THB1 is under the control of the NIT2 regulatory gene and that the hemoglobin is linked to the nitrogen assimilation pathway.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/biosíntesis , Regulación de la Expresión Génica de las Plantas/fisiología , Hemoglobinas/biosíntesis , Lisina/metabolismo , Nitrógeno/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Proteínas de Cloroplastos/química , Hemo/química , Hemo/metabolismo , Hemoglobinas/química , Hemoglobinas/genética , Concentración de Iones de Hidrógeno , Lisina/química , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Nitrógeno/químicaRESUMEN
The X-ray structures of the hemoglobin from Synechococcus sp. PCC 7002 (GlbN) were solved in the ferric bis-histidine (1.44 Å resolution) and cyanide-bound (2.25 Å resolution) states with covalently attached heme. The two structures illustrate the conformational changes and cavity opening caused by exogenous ligand binding. They also reveal an unusually distorted heme, ruffled as in c cytochromes. Comparison to the solution structure demonstrates the influence of crystal packing on several structural elements, whereas comparison to GlbN from Synechocystis sp. PCC 6803 shows subtle differences in heme geometries and environment. The new structures will be instrumental in elucidating GlbN reactivity.
Asunto(s)
Cristalografía por Rayos X/métodos , Hemo/química , Hemoglobinas/química , Resonancia Magnética Nuclear Biomolecular/métodos , Synechococcus/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions. Several TrHbs have been studied in vitro using engineered recombinant peptides. These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50-60 µL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage.