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Abstract Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Fission yeast Schizosaccharomyces pombe shares many characteristics with higher eukaryotes. Here, the authors investigate the structure and function of respiratory complex IV from S. pombe, reveal the subunit arrangements and the reaction sequence of O2 reduction.

Abstract Background Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker’s yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex. Results Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility. Conclusions Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

ABSTRACT The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+→N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

Summary: Exposure to air pollution is associated with adverse health effects, with particulate matter (PM) and ozone (O3) both indicated to be of considerable importance. Diesel engine exhaust (DE) and O3 generate substantial inflammatory effects in the airways. However, as yet it has not been determined whether a subsequent O3 exposure would add to the diesel-induced airway inflammatory effects. Healthy subjects underwent two separate exposure series: A 1-h DE exposure at a PM-concentration of 300μg/m3, followed after 5h by a 2-h exposure to filtered air and 0.2ppm O3, respectively. Induced sputum was collected 18h after the second exposure. A significant increase in the percentage of neutrophils (PMN) and concentration of myeloperoxidase (MPO) was seen in sputum post DE+O3 vs. DE+air (p<0.05 and <0.05, respectively). Significant associations were observed between the responses in MPO concentration and total PMN cells (p=0.001), and also between MPO and matrix metalloproteinase-9 (MMP-9) (p<0.001). The significant increase of PMN and MPO after the DE+O3 exposures, compared to DE+air, denotes an O3-induced magnification of the DE-induced inflammation. Furthermore, the correlation between responses in MPO and number of PMNs and MMP-9 illustrate that the PMNs are activated, resulting in a more potent inflammatory response. The present study indicates that O3 exposure adds significantly to the inflammatory response that is established by diesel exhaust. This interaction between exposure to particulate pollution and O3 in sequence should be taken into consideration when health effects of air pollution are considered. [Copyright &y& Elsevier]

The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O 2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O 2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O 2 molecule has bound to the diferrous diiron active site and before the OO bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both NN bond formation and NO bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O 2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual OO bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O 2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O 2 reduction reaction in the family of heme‑copper oxidases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc 1 ) and one of the organism's terminal oxidases, cytochrome cbb 3 , which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c 4 and a single heme cytochrome c 5 . Together, these proteins allow electron transfer from ubiquinol in cytochrome bc 1 to oxygen in cytochrome cbb 3 . We also present evidence that different isoforms of cytochrome cbb 3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc 1 - cbb 3 SCs also exist in other bacterial pathogens.

The M. smegmatis respiratory III 2 IV 2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e - /s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, Cu A , displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome c oxidase (complex IV), which receives electrons from cytochrome bc 1 (complex III), via membrane-bound or water-soluble cytochrome c . These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III 2 IV 1/2 Saccharomyces cerevisiae mitochondrial supercomplex as well as the obligate III 2 IV 2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of complex III, proton uptake by Cyt c O, as well as mechanistic and structural solutions to the electronic link between complexes III and IV.

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc 1 and aa 3 -type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O 2 reduction activity of the cyt. bc 1 -aa 3 supercomplex. The potential role of the membrane-anchored cyt. c y as a component in supercomplexes was also investigated., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The Saccharomyces cerevisiae respiratory supercomplex factor 2 (Rcf2) is a 224-residue protein located in the mitochondrial inner membrane where it is involved in the formation of supercomplexes composed of cytochrome bc 1 and cytochrome c oxidase. We previously demonstrated that Rcf2 forms a dimer in dodecylphosphocholine micelles, and here we report the solution NMR structure of this Rcf2 dimer. Each Rcf2 monomer has two soluble α helices and five putative transmembrane (TM) α helices, including an unexpectedly charged TM helix at the C terminus, which mediates dimer formation. The NOE contacts indicate the presence of inter-monomer salt bridges and hydrogen bonds at the dimer interface, which stabilize the Rcf2 dimer structure. Moreover, NMR chemical shift change mapping upon lipid titrations as well as molecular dynamics analysis reveal possible structural changes upon embedding Rcf2 into a native lipid environment. Our results contribute to the understanding of respiratory supercomplex formation and regulation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cytochrome c oxidase is a membrane-bound redox-driven proton pump that harbors two proton-transfer pathways, D and K, which are used at different stages of the reaction cycle. Here, we address the question if a D pathway with a modified energy landscape for proton transfer could take over the role of the K pathway when the latter is blocked by a mutation. Our data indicate that structural alterations near the entrance of the D pathway modulate energy barriers that influence proton transfer to the proton-loading site. The data also suggest that during reduction of the catalytic site, its protonation has to occur via the K pathway and that this proton transfer to the catalytic site cannot take place through the D pathway., (© 2019 Federation of European Biochemical Societies.)

The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes., (Copyright © 2019 Knopp et al.)

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (Fe B ). Heme b 3 and Fe B constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks Fe B to investigate the role of Fe B during catalysis. We show that in the Fe B -less cNOR, binding of both NO and O 2 to heme b 3 still occurs but further reduction is impaired, although to a lesser degree for O 2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed., (© 2019 Federation of European Biochemical Societies.)

The heme‑copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O 2 reduction and for proton pumping. This pathway, called the K C -pathway, starts at Glu-49 P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the K C -pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pK a of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily., (Copyright © 2018 Elsevier B.V. All rights reserved.)