RESUMO
HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'â5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Ferro/metabolismo , Manganês/metabolismo , Thermotoga maritima/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/química , Animais , Domínio Catalítico/fisiologia , Bovinos , Ativação Enzimática/fisiologia , Estrutura Secundária de Proteína , Soroalbumina BovinaRESUMO
This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth's nitrogen cycle where they catalyze the proton-dependent two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV-vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reaction of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent (57)Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. The outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Oxirredutases/química , Modelos Moleculares , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nitroso/química , Óxido Nitroso/metabolismo , Oxirredutases/metabolismo , Teoria Quântica , Espectrofotometria UltravioletaRESUMO
The location of the Trp radical and the catalytic function of the [Fe(IV)âO Trp191(â¢+)] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71(â¢) and Tyr236(â¢) is independent of the [Fe(IV)âO Trp191(â¢+)] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Å from the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632-1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71(â¢) as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248-255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665-11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557-8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.
Assuntos
Citocromo-c Peroxidase/metabolismo , Expectorantes/metabolismo , Guaiacol/metabolismo , Heme/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Biocatálise , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/genética , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Expectorantes/química , Guaiacol/química , Heme/química , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Propriedades de Superfície , Triptofano/química , Triptofano/metabolismo , Tirosina/químicaRESUMO
Tyrosine is a conserved redox-active amino acid that plays important roles in heme-copper oxidases (HCO). Despite the widely proposed mechanism that involves a tyrosyl radical, its direct observation under O2 reduction conditions remains elusive. Using a functional oxidase model in myoglobin called F33Y-Cu(B)Mb that contains an engineered tyrosine, we report herein direct observation of a tyrosyl radical during both reactions of H2O2 with oxidized protein and O2 with reduced protein by electron paramagnetic resonance spectroscopy, providing a firm support for the tyrosyl radical in the HCO enzymatic mechanism.
Assuntos
Peróxido de Hidrogênio/química , Mioglobina/química , Oxirredutases/química , Oxigênio/química , Tirosina/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Radicais Livres/metabolismo , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Mioglobina/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Tirosina/metabolismoRESUMO
The intracellular level of the ubiquitous bacterial secondary messenger, cyclic di-(3',5')-guanosine monophosphate (c-di-GMP), represents a balance between its biosynthesis and degradation, the latter via specific phosphodiesterases (PDEs). One class of c-di-GMP PDEs contains a characteristic HD-GYP domain. Here we report that an HD-GYP PDE from Vibrio cholerae contains a non-heme diiron-carboxylate active site, and that only the reduced form is active. An engineered D-to-A substitution in the HD dyad caused loss of c-di-GMP PDE activity and of two iron atoms. This report constitutes the first demonstration that a non-heme diiron-carboxylate active site can catalyze the c-di-GMP PDE reaction and that this activity can be redox regulated in the HD-GYP class.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/química , Proteínas de Bactérias/química , Heme/química , Ferro/química , Vibrio cholerae/enzimologia , Domínio Catalítico , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Denitrifying NO reductases are evolutionarily related to the superfamily of heme--copper terminal oxidases. These transmembrane protein complexes utilize a heme-nonheme diiron center to reduce two NO molecules to N(2)O. To understand this reaction, the diiron site has been modeled using sperm whale myoglobin as a scaffold and mutating distal residues Leu-29 and Phe-43 to histidines and Val-68 to a glutamic acid to create a nonheme Fe(B) site. The impact of incorporation of metal ions at this engineered site on the reaction of the ferrous heme with one NO was examined by UV-vis absorption, EPR, resonance Raman, and FTIR spectroscopies. UV--vis absorption and resonance Raman spectra demonstrate that the first NO molecule binds to the ferrous heme, but while the apoproteins and Cu(I)- or Zn(II)-loaded proteins show characteristic EPR signatures of S = 1/2 six-coordinate heme {FeNO}(7) species that can be observed at liquid nitrogen temperature, the Fe(II)-loaded proteins are EPR silent at ≥30 K. Vibrational modes from the heme [Fe-N-O] unit are identified in the RR and FTIR spectra using (15)NO and (15)N(18)O. The apo and Cu(I)-bound proteins exhibit ν(FeNO) and ν(NO) that are only marginally distinct from those reported for native myoglobin. However, binding of Fe(II) at the Fe(B) site shifts the heme ν(FeNO) by 17 cm(-1) and the ν(NO) by -50 cm(-1) to 1549 cm(-1). This low ν(NO) is without precedent for a six-coordinate heme {FeNO}(7) species and suggests that the NO group adopts a strong nitroxyl character stabilized by electrostatic interaction with the nearby nonheme Fe(II). Detection of a similarly low ν(NO) in the Zn(II)-loaded protein supports this interpretation.
Assuntos
Heme/metabolismo , Ferro/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Análise Espectral , Apoproteínas/química , Apoproteínas/metabolismo , Domínio Catalítico , Elétrons , Modelos Moleculares , Óxido Nítrico/metabolismo , Fotólise , Pseudomonas aeruginosa/enzimologia , Eletricidade Estática , TemperaturaRESUMO
A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe(B)Mb(-His)). A high resolution (1.65 A) crystal structure of Cu(II)-CN(-)-Fe(B)Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe(B)Mb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe(B)Mb(-His) and Fe(II)-Fe(B)Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N(2)O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe(B)Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe(B)Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.
Assuntos
Substituição de Aminoácidos , Ferro , Mioglobina/química , Mioglobina/metabolismo , Oxirredutases/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Mioglobina/genética , Conformação Proteica , Espectrofotometria UltravioletaRESUMO
A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe(B) site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by approximately 110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a approximately 100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe(B) site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.
Assuntos
Glutamatos/metabolismo , Metais/metabolismo , Mioglobina/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Engenharia de Proteínas , Substituição de Aminoácidos/genética , Animais , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Íons , Ferro/química , Ferro/metabolismo , Mioglobina/química , Óxido Nítrico/metabolismo , Oxirredução , Cachalote , Fatores de TempoRESUMO
Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem Fe(B) centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.