Combining a Commercial Mixer with a Wall-Tube Electrode Allows the Arbitrary Control of Concentrations in Protein Film Electrochemistry.

Protein film electrochemistry is a technique in which an enzyme is immobilized on an electrode in a configuration that allows following the changes in turnover frequency as a response to changes in the experimental conditions. Insights into the reactivity of the enzyme can be obtained by quantitatively modeling such responses. As a consequence, the more the technique allows flexibility in changing conditions, the more useful it becomes. The most commonly used setup, based on the rotating disc electrode, allows easy stepwise increases in the concentration of nongaseous substrates, or exposure to constant concentration of dissolved gas, but does not permit to easily decrease the concentration of nongaseous substrates, or to change the concentration of dissolved gas in a stepwise fashion. To overcome the limitation by mass transport of the substrate toward the electrode when working with fast enzymes, we have designed another kind of electrochemical cell based on the wall-tube electrode (WTE). We demonstrate here that by using a system combining two syringe pumps, a commercial mixer, and the WTE, it is possible to change the concentration of species in a stepwise fashion in all directions, opening new possibilities to study redox enzymes. As a proof of concept, this device was applied to the study of the electrochemical response of the cytochrome c nitrite reductase of Desulfovibrio desulfuricans.

Structure of a membrane-bound menaquinol:organohalide oxidoreductase.

Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the structure of a menaquinol:organohalide oxidoreductase obtained by cryo-EM. The membrane-bound protein was isolated from Desulfitobacterium hafniense strain TCE1 as a PceA2B2 complex catalysing the dechlorination of tetrachloroethene. Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. In this work, the structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria.

Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation.

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from -150 to -358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.

Genomic insight into iron acquisition by sulfate-reducing bacteria in microaerophilic environments.

Historically, sulfate-reducing bacteria (SRB) have been considered to be strict anaerobes, but reports in the past couple of decades indicate that SRB tolerate exposure to O2 and can even grow in aerophilic environments. With the transition from anaerobic to microaerophilic conditions, the uptake of Fe(III) from the environment by SRB would become important. In evaluating the metabolic capability for the uptake of iron, the genomes of 26 SRB, representing eight families, were examined. All SRB reviewed carry genes (feoA and feoB) for the ferrous uptake system to transport Fe(II) across the plasma membrane into the cytoplasm. In addition, all of the SRB genomes examined have putative genes for a canonical ABC transporter that may transport ferric siderophore or ferric chelated species from the environment. Gram-negative SRB have additional machinery to import ferric siderophores and ferric chelated species since they have the TonB system that can work alongside any of the outer membrane porins annotated in the genome. Included in this review is the discussion that SRB may use the putative siderophore uptake system to import metals other than iron.

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples.

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

Redox loops in anaerobic respiration - The role of the widespread NrfD protein family and associated dimeric redox module.

In prokaryotes, the proton or sodium motive force required for ATP synthesis is produced by respiratory complexes that present an ion-pumping mechanism or are involved in redox loops performed by membrane proteins that usually have substrate and quinone-binding sites on opposite sides of the membrane. Some respiratory complexes include a dimeric redox module composed of a quinone-interacting membrane protein of the NrfD family and an iron­sulfur protein of the NrfC family. The QrcABCD complex of sulfate reducers, which includes the QrcCD module homologous to NrfCD, was recently shown to perform electrogenic quinone reduction providing the first conclusive evidence for energy conservation among this family. Similar redox modules are present in multiple respiratory complexes, which can be associated with electroneutral, energy-driven or electrogenic reactions. This work discusses the presence of the NrfCD/PsrBC dimeric redox module in different bioenergetics contexts and its role in prokaryotic energy conservation mechanisms.

An electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation.

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H+/e- ratio of 1. Although the complex does not act as a H+-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts.

Electron transfer between the QmoABC membrane complex and adenosine 5'-phosphosulfate reductase.

The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5'-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB.

Steady-state kinetics with nitric oxide reductase (NOR): new considerations on substrate inhibition profile and catalytic mechanism.

Nitric oxide reductase (NOR) from denitrifying bacteria is an integral membrane protein that catalyses the two electron reduction of NO to N2O, as part of the denitrification process, being responsible for an exclusive reaction, the NN bond formation, the key step of this metabolic pathway. Additionally, this class of enzymes also presents residual oxidoreductase activity, reducing O2 to H2O in a four electron/proton reaction. In this work we report, for the first time, steady-state kinetics with the Pseudomonas nautica NOR, either in the presence of its physiological electron donor (cyt. c552) or immobilised on a graphite electrode surface, in the presence of its known substrates, namely NO or O2. The obtained results show that the enzyme has high affinity for its natural substrate, NO, and different kinetic profiles according to the electron donor used. The kinetic data, as shown by the pH dependence, is modelled by ionisable amino acid residues nearby the di-nuclear catalytic site. The catalytic mechanism is revised and a mononitrosyl-non-heme Fe complex (FeB(II)-NO) species is favoured as the first catalytic intermediate involved on the NO reduction.

Electrochemical behaviour of bacterial nitric oxide reductase-evidence of low redox potential non-heme Fe(B) gives new perspectives on the catalytic mechanism.

Nitric oxide reductase (NOR) is a membrane bound enzyme involved in the metabolic denitrification pathway, reducing nitric oxide (NO) to nitrous oxide (N(2)O), subsequently promoting the formation of the NN bond. Three types of bacterial NOR are known, namely cNOR, qNOR and qCuNOR, that differ on the physiological electron donor. cNOR has been purified as a two subunit complex, the NorC, anchored to the cytoplasmic membrane, with a low-spin heme c, and the NorB subunit showing high structural homology with the HCuO subunit I, comprising a bis-histidine low-spin heme b and a binuclear iron centre. The binuclear iron centre is the catalytic site and it is formed by a heme b(3) coupled to a non-heme iron (Fe(B)) through a µ-oxo bridge. The catalytic mechanism is still under discussion and three hypotheses have been proposed: the trans-mechanism, the cis-Fe(B) and the cis-heme b(3) mechanisms. In the present work, the Pseudomonas nautica cNOR electrochemical behaviour was studied by cyclic voltammetry (CV), using a pyrolytic graphite electrode modified with the immobilised protein. The protein redox centres were observed and the formal redox potentials were determined. The binuclear iron centre presents the lowest redox potential value, and discrimination between the heme b(3) and Fe(B) redox processes was attained. Also, the number of electrons involved and correspondent surface electronic transfer rate constants were estimated. The pH dependence of the observed redox processes was determined and some new insights on the NOR catalytic mechanism are discussed.

Spectroscopic evidence for and characterization of a trinuclear ferroxidase center in bacterial ferritin from Desulfovibrio vulgaris Hildenborough.

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a µ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica.

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

Rubredoxin mutant A51C unfolding dynamics: a Förster Resonance Energy Transfer study.

The unfolding dynamics of the rubredoxin mutant A51C (RdA51C) from Desulfovibrio vulgaris (DvRd) was studied on the temperature range from 25 degrees C to 90 degrees C and by incubation at 90 degrees C. By Förster Resonance Energy Transfer (FRET) the donor (D; Trp37) to acceptor (A; 1,5-IAEDANS) distance distribution was probed at several temperatures between 25 degrees C and 90 degrees C, and incubation times at 90 degrees C. From 25 degrees C to 50 degrees C the half-width distributions values (hw) are small and the presence of a discrete D-A distance was considered. At temperatures higher than 60 degrees C broader hw values were observed reflecting the existence of a distance distribution. The protein denaturation was only achieved by heating the solution for 2h at 90 degrees C, as probed by the increase of the D-A mean distance. From Trp fluorescence it was shown that its vicinity was maintained until approximately 70 degrees C, being the protein hydrodynamic radius invariant until 50 degrees C. However, at approximately 70 degrees C a change in the partial unfolding kinetics indicates the disruption of specific H-bonds occurring in the hydrophobic core. The red shift of 13nm, observed on the Trp37 emission, confirms the exposition of Trp to solvent after protein incubation at 90 degrees C for 2.5h.

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas.

The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12 kDa protein that contains a novel mixed-metal sulfide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25 A resolution in-house and to beyond 2.0 A resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106 A.

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

We 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.