Source and reduction of nitrous oxide

PTDC/BBB-BQB/0129/2014. UID/QUI/50006/2019. UlD/Multi/04378/2019. MSPC acknowledges FCT/MCTES for funding her "Research Position" (signed with FCT NOVA in accordance with DL.57/2016 and Lei 57/2017). Sem PDF conforme despacho.Nitrous oxide is a potent greenhouse gas with a global warming impact 300-fold higher than carbon dioxide. Due to its exponential increase in the atmosphere and its implications in climate change there is the need to develop strategies to mitigate its emissions and to reduce it to the inert dinitrogen gas. Only three enzymes have been reported to be able to reduce nitrous oxide, namely nitrogenase, one multicopper oxidase and nitrous oxide reductase, with the latter being the only one with a relevant physiological activity. In this enzyme, reduction of nitrous oxide occurs in a unique catalytic tetranuclear sulfide center, named “CuZ” center, a complex center required to overcome the high activation barrier of this reaction. Nitrous oxide reductase can be isolated with “CuZ” center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), that differ in their catalytic and spectroscopic properties. Recently, another step towards a better understanding of the catalytic and activation mechanism of this enzyme was taken by identifying and spectroscopically characterizing an intermediate species of its catalytic cycle, CuZ 0 . A different approach for N 2 O reduction can be attained using model compounds. The unique structural motif present in “CuZ” center, a Cu 4 (µ 4 -S), has been a challenge for inorganic synthesis but several synthetic clusters that mimic different forms of “CuZ” center have been reported. Model compounds for the oxidation states involved in N 2 O reduction are also available. The advances in this area will be discussed in light of the recent data, with structural and functional model compounds of N 2 OR active site.authorsversionpublishe

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Biomol NMR Assign (2007) 1:81–83 DOI 10.1007/s12104-007-9022-3We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo–Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments

Molybdenum Induces the expression of a protein containing a new heterometallic Mo-Fe cluster in desulfoVibrio alaskensis

Biochemistry. 2009 Feb 10;48(5):873-82. doi: 10.1021/bi801773t.The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed

Incorporation of a molybdenum atom in a Rubredoxin-type Centre of a de novo-designed α3DIV-L21C three-helical bundle peptide

PB would thank the PTNMRPhD (PD/00065/2013). VLP thanks the NIH for support (GM141086).The rational design and functionalization of small, simple, and stable peptides scaffolds is an attractive avenue to mimic catalytic metal-centres of complex proteins, relevant for the design of metalloenzymes with environmental, biotechnological and health impacts. The de novo designed α3DIV-L21C framework has a rubredoxin-like metal binding site and was used in this work to incorporate a Mo-atom. Thermostability studies using differential scanning calorimetry showed an increase of 4 °C in the melting temperature of the Mo-α3DIV-L21C when compared to the apo-α3DIV-L21C. Circular dichroism in the visible and far-UV regions corroborated these results showing that Mo incorporation provides stability to the peptide even though there were almost no differences observed in the secondary structure. A formal reduction potential of ∼ −408 mV vs. NHE, pH 7.6 was determined. Combining electrochemical results, EPR and UV–visible data we discuss the oxidation state of the molybdenum centre in Mo-α3DIV-L21C and propose that is mainly in a Mo (VI) oxidation state.publishersversionpublishe

The heme-based oxygen sensor Rhizobium etli FixL: influence of auxiliary ligands on heme redox potential and implications on the enzyme activity

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the Fe redox couple of the heme domain. In this work, midpoint potentials (E) assigned to the Fe redox couple of the heme domain of FixL from Rhizobium etli (ReFixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded ReFixL protein (+ 19 mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. E = − 51, − 57 and − 156 mV for O, imidazole and CN, respectively. Upon binding to CO, which does not affect the kinase active, E was observed at + 21 mV. The potential values observed for Fe of the heme domain of ReFixL upon binding to CO and O do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the Fe state. Our results suggest that the conformational changes that switch off kinase activity upon O binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the Fe state and counterbalances the Fe-stabilizing influence of the O ligand

The Tetranuclear Copper-Sulfide Center of Nitrous Oxide Reductase

Nitrous oxide reductase catalyzes the reduction of nitrous oxide (N2O) to dinitrogen (N2) and water at a catalytic tetranuclear copper sulfide center, named CuZ, overcoming the high activation energy of this reaction. In this center each Cu atom is coordinated by two imidazole rings of histidine side-chains, with the exception of one named CuIV. This enzyme has been isolated with CuZ in two forms CuZ(4Cu1S) and CuZ(4Cu2S), which differ in the CuI-CuIV bridging ligand, leading to considerable differences in their spectroscopic and catalytic properties. The Cu atoms in CuZ(4Cu1S) can be reduced to the [4Cu1+] oxidation state, and its catalytic properties are compatible with the nitrous oxide reduction rates of whole cells, while in CuZ(4Cu2S) they can only be reduced to the [1Cu2C-3Cu1C] oxidation state, which has a very low turnover number. The catalytic cycle of this enzyme has been explored and one of the intermediates, CuZ0, has recently been identified and shown to be in the [1Cu2+-3Cu1+] oxidation state. Contrary to CuZ(4Cu2S), CuZ0 is rapidly reduced intramolecularly by the electron transferring center of the enzyme, CuA, to [4Cu1+] by a physiologically relevant redox partner. The three-dimensional structure of nitrous oxide reductase with the CuZ center either as CuZ(4Cu1S) or as CuZ(4Cu2S) shows that it is a unique functional dimer, with the CuZ of one subunit receiving electrons from CuA of the other subunit. The complex nature of this center has posed some questions relative to its assembly, which are only partially answered, as well as to which is the active form of CuZ in vivo. The structural, spectroscopic, and catalytic features of the two forms of CuZ will be addressed here, as well as its assembly. The understanding of its catalytic features, activation, and assembly is essential to develop strategies to decrease the release of nitrous oxide to the atmosphere and to reduce its concentration in the stratosphere, as well as to serve as inspiration to synthetic inorganic chemists to develop new models of this peculiar and challenging copper sulfide center.publishersversionpublishe

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe–4S] and one [4Fe–4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S]+ 1/0 (E0′ = − 158 ± 5 mV); [4Fe–4S]+ 2/+1 (E0′ = − 474 ± 5 mV) and [3Fe–4S]0/− 2 (E0′ = − 660 ± 5 mV). The effect of pH on these signals showed that the reduced [3Fe–4S]0 cluster has a pKʹred′ = 5.1 ± 0.1, the [4Fe–4S]+ 2/+1 centre is pH independent, and the [3Fe–4S]0/−2 reduction is accompanied by the binding of two protons. The ability of the [3Fe–4S]0 cluster to be converted into a new [4Fe–4S] cluster was proven. The redox potential of the original [4Fe–4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70 mV) of the redox potential of the original centre. Being most [Fe–S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.Fil: Grazina, Raquel. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; PortugalFil: Sousa, Patrícia M. Paes de. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; PortugalFil: Brondino, Carlos Dante. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe; ArgentinaFil: Carepo, Marta S. P.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; PortugalFil: Moura, Isabel. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; PortugalFil: Moura, José J. G.. Universidade Nova de Lisboa. Faculdade de Ciências e Tecnologia. Departamento de Química. REQUIMTE/CQFB; Portuga

Ligand accessibility to heme cytochrome b 5 coordinating sphere and enzymatic activity enhancement upon tyrosine ionization

FCT/MCTES (UID/QUI/50006/2019) FCT/MCTES post-doctoral grant (SFRH/BPD/100069/2014)Recently, we observed that at extreme alkaline pH, cytochrome b 5 (Cb 5 ) acquires a peroxidase-like activity upon formation of a low spin hemichrome associated with a non-native state. A functional characterization of Cb 5 , in a wide pH range, shows that oxygenase/peroxidase activities are stimulated in alkaline media, and a correlation between tyrosine ionization and the attained enzymatic activities was noticed, associated with an altered heme spin state, when compared to acidic pH values at which the heme group is released. In these conditions, a competitive assay between imidazole binding and Cb 5 endogenous heme ligands revealed the appearance of a binding site for this exogenous ligand that promotes a heme group exposure to the solvent upon ligation. Our results shed light on the mechanism behind Cb 5 oxygenase/peroxidase activity stimulation in alkaline media and reveal a role of tyrosinate anion enhancing Cb 5 enzymatic activities on the distorted protein before maximum protein unfolding.authorsversionpublishe

Reconstruction of the Fatty Acid Biosynthetic Pathway of Exiguobacterium antarcticum B7 Based on Genomic and Bibliomic Data

Exiguobacterium antarcticum B7 is extremophile Gram-positive bacteria able to survive in cold environments. A key factor to understanding cold adaptation processes is related to the modification of fatty acids composing the cell membranes of psychrotrophic bacteria. In our study we show the in silico reconstruction of the fatty acid biosynthesis pathway of E. antarcticum B7. To build the stoichiometric model, a semiautomatic procedure was applied, which integrates genome information using KEGG and RAST/SEED. Constraint-based methods, namely, Flux Balance Analysis (FBA) and elementary modes (EM), were applied. FBA was implemented in the sense of hexadecenoic acid production maximization. To evaluate the influence of the gene expression in the fluxome analysis, FBA was also calculated using the log2⁡FC values obtained in the transcriptome analysis at 0°C and 37°C. The fatty acid biosynthesis pathway showed a total of 13 elementary flux modes, four of which showed routes for the production of hexadecenoic acid. The reconstructed pathway demonstrated the capacity of E. antarcticum B7 to de novo produce fatty acid molecules. Under the influence of the transcriptome, the fluxome was altered, promoting the production of short-chain fatty acids. The calculated models contribute to better understanding of the bacterial adaptation at cold environments

Orange protein from Desulfovibrio alaskensis G20: insights into the Mo-Cu cluster protein-assisted synthesis

International audienceA novel metalloprotein containing a unique [S2MoS2CuS2MoS2](3-) cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 +/- A 0.1 Da by ESI-MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S-Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV-visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde\₃198) increased 6.6 +/- A 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria