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

A [4Fe-4S]-Fe(CO)(CN)-L-cysteine intermediate is the first organometallic precursor in [FeFe] hydrogenase H-cluster bioassembly.

Biosynthesis of the [FeFe] hydrogenase active site (the 'H-cluster') requires the interplay of multiple proteins and small molecules. Among them, the radical S-adenosylmethionine enzyme HydG, a tyrosine lyase, has been proposed to generate a complex that contains an Fe(CO)2(CN) moiety that is eventually incorporated into the H-cluster. Here we describe the characterization of an intermediate in the HydG reaction: a [4Fe-4S][(Cys)Fe(CO)(CN)] species, 'Complex A', in which a CO, a CN- and a cysteine (Cys) molecule bind to the unique 'dangler' Fe site of the auxiliary [5Fe-4S] cluster of HydG. The identification of this intermediate-the first organometallic precursor to the H-cluster-validates the previously hypothesized HydG reaction cycle and provides a basis for elucidating the biosynthetic origin of other moieties of the H-cluster

Omics profiles used to evaluate the gene expression of Exiguobacterium antarcticum B7 during cold adaptation

Abstract\ud \ud Background\ud \ud Exiguobacterium antarcticum strain B7 is a Gram-positive psychrotrophic bacterial species isolated in Antarctica. Although this bacteria has been poorly studied, its genome has already been sequenced. Therefore, it is an appropriate model for the study of thermal adaptation. In the present study, we analyzed the transcriptomes and proteomes of E. antarcticum B7 grown at 0°C and 37°C by SOLiD RNA-Seq, Ion Torrent RNA-Seq and two-dimensional difference gel electrophoresis tandem mass spectrometry (2D-DIGE-MS/MS).\ud \ud \ud Results\ud We found expression of 2,058 transcripts in all replicates from both platforms and differential expression of 564 genes (absolute log2FC ≥1, P-value <0.001) comparing the two temperatures by RNA-Seq. A total of 73 spots were differentially expressed between the two temperatures on 2D-DIGE, 25 of which were identified by MS/MS. Some proteins exhibited patterns of dispersion in the gel that are characteristic of post-translational modifications.\ud \ud \ud Conclusions\ud Our findings suggest that the two sequencing platforms yielded similar results and that different omic approaches may be used to improve the understanding of gene expression. To adapt to low temperatures, E. antarcticum B7 expresses four of the six cold-shock proteins present in its genome. The cold-shock proteins were the most abundant in the bacterial proteome at 0°C. Some of the differentially expressed genes are required to preserve transcription and translation, while others encode proteins that contribute to the maintenance of the intracellular environment and appropriate protein folding. The results denote the complexity intrinsic to the adaptation of psychrotrophic organisms to cold environments and are based on two omic approaches. They also unveil the lifestyle of a bacterial species isolated in Antarctica.CNPqCAPESUFPAFINEPFAPEMIGFundação para a Ciência e a Tecnologia de Portugal (FCT

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

Exposure to an extremely low-frequency electromagnetic field only slightly modifies the proteome of Chromobacterium violaceum ATCC 12472

Several studies of the physiological responses of different organisms exposed to extremely low-frequency electromagnetic fields (ELF-EMF) have been described. In this work, we report the minimal effects of in situ exposure to ELF-EMF on the global protein expression of Chromobacterium violaceum using a gel-based proteomic approach. The protein expression profile was only slightly altered, with five differentially expressed proteins detected in the exposed cultures; two of these proteins (DNA-binding stress protein, Dps, and alcohol dehydrogenase) were identified by MS/MS. The enhanced expression of Dps possibly helped to prevent physical damage to DNA. Although small, the changes in protein expression observed here were probably beneficial in helping the bacteria to adapt to the stress generated by the electromagnetic field