Visible and UV-resonance Raman spectroscopy was employed to investigate the reaction of NO with cytochrome
caa
3
from
Thermus thermophilus
. We show the formation of the hyponitrite (HO-N&z.dbd;N-O)
...−
bound to the heme
a
3
species (
ν
N&z.dbd;N
= 1330 cm
−1
) forming a high spin complex in the oxidized heme
a
3
Fe/Cu
B
binuclear center of
caa
3
-oxidoreductase. In the absence of heme
a
3
Fe
2+
-NO formation, the electron required for the formation of the N&z.dbd;N bond originates from the autoreduction of Cu
B
by NO, producing nitrite. With the identification of the hyponitrite intermediate the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification is fully supported and the mechanism for the 2e
−
/2H
+
reduction of NO to N
2
O can be described with more certainty.
We present UV-Raman evidence for the formation of the hyponitrite (HO-N&z.dbd;N-O
−
) species in the binuclear heme
a
3
Fe-Cu
B
center (
ν
N-N = 1330 cm
−1
) of
caa
3
heme-copper oxidoreductase from
Thermus thermophilus.
Here, we report the growth of bioelectret crystal films of L-leucine on conductive substrates and detail the first quantitative measurements of the direct piezoelectric effect in this proteinogenic ...amino acid. Through extensive electromechanical characterisation, we demonstrate that L-leucine is a promising candidate material for use in non-toxic, biocompatible and biodegradable energy harvesting and sensing devices. The data presented here is among, if not the, largest set of quasi-static, longitudinal piezoelectric measurements on crystalline films of a proteinogenic amino acid to date. We substantiate these measurements using a combination of density functional theory calculations and optical microscopy. Our data provides a further example of the enormous unexploited potential of amino acids and similar biological materials in flexible energy harvesting applications. Their combination of sizeable piezoelectric strain constants and extremely low elastic and dielectric constants makes them ideal as biocompatible replacements for toxic, expensive inorganic piezoelectric materials.
Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They ...catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)
), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDH
from
, as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles. In this protocol, we describe ALDH
heterologous expression in
, purification, and activity assay (based on Shortall
, 2021 ). ALDH
was first copurified as a contaminant during caa
-type cytochrome oxidase isolation from
. This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15-20 mg/L ALDH
. For purification of the thermophilic his-tagged ALDH
, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH).
Flow chart outlining the steps in ALDH
expression and purification, highlighting the approximate time required for each step.
Time-resolved step-scan FTIR spectroscopy has been employed to probe the dynamics of the
₃ oxidoreductase from
in the ns-μs time range and in the pH/pD 6-9 range. The data revealed a pH/pD ...sensitivity of the D372 residue and of the ring-A propionate of heme
₃. Based on the observed transient changes a model in which the protonic connectivity of w941-w946-927 to the D372 and the ring-A propionate of heme
₃ is described.
Cytochrome c oxidase is a respiratory enzyme catalysing the energy‐conserving reduction of molecular oxygen to water. The crystal structure of the ba3‐cytochrome c oxidase from Thermus thermophilus ...has been determined to 2.4 Å resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure‐based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from CuA to CuB. Specific features of the ba3‐oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O2−, OH− or H2O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.
Cytochrome aa 3 from Paracoccus denitrificans and cytochrome ba 3 from Thermus thermophilus, two distinct members of the heme–copper oxidase superfamily, were immobilized on electrodes modified with ...gold nanoparticles. This procedure allowed us to achieve direct electron transfer between the enzyme and the gold nanoparticles and to obtain evidence for different electrocatalytic properties of the two enzymes. The pH dependence and thermostability reveal that the enzymes are highly adapted to their native environments. These results suggest that evolution resulted in different solutions to the common problem of electron transfer to oxygen.
NO laughing matter: The enzymatic production of laughing gas (N2O) from two molecules of NO requires the formation of the NN bond and the subsequent cleavage of the NOH bond. Resonance Raman ...spectroscopic studies have now provided evidence for the formation of the key intermediate, the hyponitrite species (see the optimized molecular structure), in the heme a3 FeCuB binuclear center of ba3‐oxidoreductase from Thermus thermophilus.
Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we ...compared the kinetics of dioxygen reduction by ba₃ cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile O₂-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the O₂-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba₃ simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which O₂ binding to heme a₃ at 90 μM O₂ occurs with lifetimes of 9.3 and 110 μs in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 μs), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 x 10⁹ M⁻¹ s⁻¹ for O₂ binding to ba₃ in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The O₂ bond cleavage in ba₃ of 4.8 μs is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of O₂ to the active site in ba₃ and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a₃ site in ba₃, making the CO flow-flash method inapplicable.
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•Nanoboron particles were characterized in textile coating solutions for stability.•Nanoboron coating changes surface nature and antimicrobial response on textiles.•Boron ...nanoparticles can limit the bacterial growth of both Gram (−) and (+) species.•Bacteria are more prone to stable nanoboron, which can pass through cell walls.•Strong attachment of nanoboron on textile surface limits antibacterial efficacy.
The antibacterial properties of boron-containing compounds are well known although there are limited studies available on the pure boron nanoparticles. In this paper, nanoboron particles are characterized in terms of their particle size, shape, stability and surface charge before and after their application onto textile surfaces to study their impact on bacterial activity. It was observed that the boron nanoparticles are effective in limiting the bacterial growth of both Gram-negative and positive species without requiring any stimulation to initiate the antibacterial action. In addition to the antibacterial functionality evaluation of the free boron nanoparticles, nanoboron coated textiles were also characterized and determined to change the wettability and surface charge of the textiles with a variable antimicrobial response to the different species. Consequently, we propose pure nanoboron as a new anti-bacterial agent that can function without external stimulation.
Decades have passed without approval of a new antibiotic class. Several companies have recently halted related discovery efforts because of multiple obstacles. One promising route under research is ...to target the lipoprotein maturation pathway in light of major recent findings and the virulence roles of lipoproteins. To support the future design of selective drugs, considerations and priority-setting are established for the main lipoprotein processing enzymes (Lgt, LspA, and Lnt) based on microbiology, biochemistry, structural biology, chemical design, and pharmacology. Although not all bacterial species will be similarly impacted by drug candidates, several advantages make LspA a top target to pursue in the development of novel antibiotics effective against bacteria that are resistant to existing drugs.
The characteristics of the lipoprotein processing enzymes Lgt, LspA, and Lnt imply lipophilic, membrane-penetrating antibiotic designs, of which the pharmacological qualities for administration must be evaluated cautiously.Each enzyme varies in relevance across bacterial species, some of which also possess multiple genes and/or functional homologs. Other enzymes and lipoprotein forms may also exist.Structural comparison of Lgt with other families (PgpB and PlsY) indicates unique insights into inhibition and the lipid-binding cleft.The mechanism of binding of the LspA inhibitor globomycin requires further studies. Several hypotheses are provided on its inhibition type and interactions with the catalytic site and protein in general.A novel LspA inhibitor, 'inhibitor-99', shares similarity with known binders of various proteins.Lnt has four catalytic residues, to which substrate access is controlled by a unique lid-loop.There is an additional pocket in Lnt for binding and stabilization that lies above the catalytic residues: lessons learned from other protein families (DHHC20 and PlsC).
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