Intrinsic electronic conductivity of individual atomically resolved amyloid crystals reveals micrometer-long hole hopping via tyrosines.

Proteins are commonly known to transfer electrons over distances limited to a few nanometers. However, many biological processes require electron transport over far longer distances. For example, soil and sediment bacteria transport electrons, over hundreds of micrometers to even centimeters, via putative filamentous proteins rich in aromatic residues. However, measurements of true protein conductivity have been hampered by artifacts due to large contact resistances between proteins and electrodes. Using individual amyloid protein crystals with atomic-resolution structures as a model system, we perform contact-free measurements of intrinsic electronic conductivity using a four-electrode approach. We find hole transport through micrometer-long stacked tyrosines at physiologically relevant potentials. Notably, the transport rate through tyrosines (105 s-1) is comparable to cytochromes. Our studies therefore show that amyloid proteins can efficiently transport charges, under ordinary thermal conditions, without any need for redox-active metal cofactors, large driving force, or photosensitizers to generate a high oxidation state for charge injection. By measuring conductivity as a function of molecular length, voltage, and temperature, while eliminating the dominant contribution of contact resistances, we show that a multistep hopping mechanism (composed of multiple tunneling steps), not single-step tunneling, explains the measured conductivity. Combined experimental and computational studies reveal that proton-coupled electron transfer confers conductivity; both the energetics of the proton acceptor, a neighboring glutamine, and its proximity to tyrosine influence the hole transport rate through a proton rocking mechanism. Surprisingly, conductivity increases 200-fold upon cooling due to higher availability of the proton acceptor by increased hydrogen bonding.

Syntrophic growth with direct interspecies electron transfer between pili-free Geobacter species.

Direct interspecies electron transfer (DIET) may prevail in microbial communities that show methanogenesis and anaerobic methane oxidation and can be an electron source to support anaerobic photosynthesis. Previous mutagenic studies on cocultures of defined Geobacter species indicate that both conductive pili and extracellular cytochromes are essential for DIET. However, the actual functional role of the pili in DIET is uncertain, as the pilus mutation strategy used in these studies affected the extracellular cytochrome profile. Here we repressed the function of pili by deleting the pilus polymerization motor PilB in both Geobacter species. The PilB mutation inhibited the pilus assembly but did not alter the pattern of extracellular cytochromes. We report that the two pilus-free Geobacter species can form aggregates and grow syntrophically with DIET. The results demonstrate that the Gmet_2896 cytochrome of Geobacter metallireducens plays a key role in DIET and that conductive pili are not necessary to facilitate DIET in cocultures of Geobacter species, and they suggest cytochromes by themselves can meditate DIET, deepening the understanding of DIET.

Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to ß-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to ß-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

Virulence factors enhance Citrobacter rodentium expansion through aerobic respiration.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonic crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonic crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygenation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the γ-secretase inhibitor dibenzazepine to diminish Notch-driven colonic crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime.

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the γ-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.

Decreasing electron flux through the cytochrome and/or alternative respiratory pathways triggers common and distinct cellular responses dependent on growth conditions.

Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the perturbation of mitochondrial function under different growth conditions.

Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.

Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage.

Long-range electron transport to Fe(III) oxide via pili with metallic-like conductivity.

The mechanisms for Fe(III) oxide reduction by Geobacter species are of interest because Geobacter species have been shown to play an important role in Fe(III) oxide reduction in a diversity of environments in which Fe(III) reduction is a geochemically significant process. Geobacter species specifically express pili during growth on Fe(III) oxide compared with growth on soluble chelated Fe(III), and mutants that cannot produce pili are unable to effectively reduce Fe(III) oxide. The pili of Geobacter sulfurreducens are electrically conductive along their length under physiologically relevant conditions and exhibit a metallic-like conductivity similar to that observed previously in synthetic organic metals. Metallic-like conductivity in a biological protein filament is a previously unrecognized mechanism for electron transport that differs significantly from the more well-known biological strategy of electron hopping/tunnelling between closely spaced redox-active proteins. The multihaem c-type cytochrome OmcS is specifically associated with pili and is necessary for Fe(III) oxide reduction. However, multiple lines of evidence, including the metallic-like conductivity of the pili and the fact that OmcS molecules are spaced too far apart for electron hopping/tunnelling, indicate that OmcS is not responsible for long-range electron conduction along the pili. The role of OmcS may be to facilitate electron transfer from the pili to Fe(III) oxide. Long-range electron transport via pili with metallic-like conductivity is a paradigm shift that has important implications not only for Fe(III) oxide reduction, but also for interspecies electron exchange in syntrophic microbial communities as well as microbe-electrode interactions and the emerging field of bioelectronics.

Spectroelectrochemical analyses of electroactive microbial biofilms.

Understanding the mechanism of ET (electron transfer) through electroactive microbial biofilms is a challenge in the field of fundamental and applied life sciences. To date, electrochemical techniques such as CV (cyclic voltammetry) have been applied successfully to study the ET process in intact microbial biofilms on electrodes, providing important insight into their redox properties. However, CV as such does not provide any structural information about the species involved in the redox process. This shortcoming may limit the understanding of the ET process in microbial biofilms. To overcome this restriction, spectroelectrochemical techniques have been designed consisting of a spectroscopic technique performed in combination with electrochemical methods on the same electrode sample. These analytical approaches allow in vivo measurements of microbial biofilms under physiologically relevant conditions and controlled applied potential. The present review describes these spectroelectrochemical methodologies and critically addresses their impact on the understanding of the ET through biofilms.

Electrochemical communication between microbial cells and electrodes via osmium redox systems.

Electrochemical communication between micro-organisms and electrodes is the integral and fundamental part of BESs (bioelectrochemical systems). The immobilization of bacterial cells on the electrode and ensuring efficient electron transfer to the electrode via a mediator are decisive features of mediated electrochemical biosensors. Notably, mediator-based systems are essential to extract electrons from the non-exoelectrogens, a major group of microbes in Nature. The advantage of using polymeric mediators over diffusible mediators led to the design of osmium redox polymers. Their successful use in enzyme-based biosensors and BFCs (biofuel cells) paved the way for exploring their use in microbial BESs. The present mini-review focuses on osmium-bound redox systems used to date in microbial BESs and their role in shuttling electrons from viable microbial cells to electrodes.

Response of mitochondrial thioredoxin PsTrxo1, antioxidant enzymes, and respiration to salinity in pea (Pisum sativum L.) leaves.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

d(1) haem biogenesis - assessing the roles of three nir gene products.

The synthesis of the modified tetrapyrrole known as d(1) haem requires several dedicated proteins which are coded for by a set of genes that are often found adjacent to the structural gene, nirS, for cytochrome cd(1) nitrite reductase. NirE, the product of the first gene in the nir biogenesis operon, was anticipated to catalyse the conversion of uroporphyrinogen III into precorrin-2; this was confirmed, but it was shown that this enzyme is less sensitive to product inhibition than similar enzymes that function in other biosynthetic pathways. Sequence analysis suggesting that one of these proteins, NirN, is a c-type cytochrome, and has similarity to the part of cytochrome cd(1) that binds d(1), was validated by recombinant production and characterization of NirN. A NirN-d(1) haem complex was demonstrated to release the cofactor to a semi-apo form of cytochrome cd(1) from which d(1) was extracted, suggesting a role for NirN in the assembly of cytochrome cd(1) (NirS). However, inactivation of nirN surprisingly led to only a marginal attenuation of growth of Paracoccus pantotrophus under anaerobic denitrifying conditions. As predicted, NirC is a c-type cytochrome; it was shown in vitro to be an electron donor to the NirN-d(1) complex.

Incorporation of respiratory cytochromes in liposomes: an efficient strategy to study the respiratory chain.

The electron transport system present in the photosynthetic apparatus and respiratory chain were evolutionary acquisitions that allowed the organisms to convert electromagnetic energy in chemical energy and thus improve the use of energy fuels. These systems acquired by prokaryotes were preserved in the highly complex organisms always with the participation of the cytochromes. Since the discovery of the cytochromes, and the isolation and association of these proteins to model membranes, the liposomes, have been used to investigative strategies to characterize the structure and function of these proteins. From these studies important findings have contributed to the comprehension of the energy transduction mechanisms and the role played by the nonredox subunits present in the protein complexes of the respiratory chain of eukaryotes.

Central role of mitochondria in metabolic regulation of liver pathophysiology.

Mitochondria play a central role in cellular energy metabolism. Oxidative phosphorylation occurs in the electron transport system of the inner mitochondrial membrane. Cytochrome aa3, b and c1 are encoded by mitochondrial DNA whereas cytochrome c is encoded by the nuclear gene, and these mitochondrial-DNA dependent cytochromes are decreased and electron transport at complex II, III and IV is disturbed in liver carcinomas and during carcinogenesis. The more the decreased cytochrome and oxidase activity are seen, the more significant is the increase in reactive oxygen species (ROS) production. ROS produced in mitochondria may be the main cause of nuclear-gene mutation in carcinogenesis. The mitochondrial dysfunction and overproduction of ROS plays a key role in progression of chronic hepatitis C and ethanol-induced liver injury. Ethanol also causes bacterial translocation in the intestine and the resulting lipopolysaccharides (LPS) activates Kupffer cells to produce pro-inflammatory cytokines. We suspect that non-alcoholic steatohepatitis (NASH) also is the result of increased ROS production in Kupffer cells and hepatocytes.

Respiration of Escherichia coli in the mouse intestine.

Mammals are aerobes that harbor an intestinal ecosystem dominated by large numbers of anaerobic microorganisms. However, the role of oxygen in the intestinal ecosystem is largely unexplored. We used systematic mutational analysis to determine the role of respiratory metabolism in the streptomycin-treated mouse model of intestinal colonization. Here we provide evidence that aerobic respiration is required for commensal and pathogenic Escherichia coli to colonize mice. Our results showed that mutants lacking ATP synthase, which is required for all respiratory energy-conserving metabolism, were eliminated by competition with respiratory-competent wild-type strains. Mutants lacking the high-affinity cytochrome bd oxidase, which is used when oxygen tensions are low, also failed to colonize. However, the low-affinity cytochrome bo(3) oxidase, which is used when oxygen tension is high, was found not to be necessary for colonization. Mutants lacking either nitrate reductase or fumarate reductase also had major colonization defects. The results showed that the entire E. coli population was dependent on both microaerobic and anaerobic respiration, consistent with the hypothesis that the E. coli niche is alternately microaerobic and anaerobic, rather than static. The results indicate that success of the facultative anaerobes in the intestine depends on their respiratory flexibility. Despite competition for relatively scarce carbon sources, the energy efficiency provided by respiration may contribute to the widespread distribution (i.e., success) of E. coli strains as commensal inhabitants of the mammalian intestine.

Plants at high altitude exhibit higher component of alternative respiration.

Total respiration, capacities of cytochrome (CytR) and alternative respiration (AR) were studied in two varieties of barley (Horedum vulgare) and wheat (Triticum aestivum) each and one variety of pea (Pisum sativum) at low (Palampur; 1300 m) and high altitudes (Kibber; 4200 m). Similar studies were carried out in naturally growing Rumex nepalensis and Trifoilum repenses at Palampur, Palchan (2250 m) and Marhi (3250 m). All the plants species exhibited lower CytR but significantly higher AR capacity at high altitude (HA) (72-1117% higher) as compared to those at low altitude (LA). Glycolytic product, pyruvate and tricarboxylic acid cycle intermediate, citrate increased with increase in altitude. While the role of these metabolites in relation to HA biology is discussed, significantly higher AR at HA is proposed to be an adaptive mechanism against the metabolic perturbations wherein it might act to lower reactive oxygen species and also provides metabolic homeostasis to plants under the environment of HA.

Oxygen reactivity of both respiratory oxidases in Campylobacter jejuni: the cydAB genes encode a cyanide-resistant, low-affinity oxidase that is not of the cytochrome bd type.

The microaerophilic bacterium Campylobacter jejuni is a significant food-borne pathogen and is predicted to possess two terminal respiratory oxidases with unknown properties. Inspection of the genome reveals an operon (cydAB) apparently encoding a cytochrome bd-like oxidase homologous to oxidases in Escherichia coli and Azotobacter vinelandii. However, C. jejuni cells lacked all spectral signals characteristic of the high-spin hemes b and d of these oxidases. Mutation of the cydAB operon of C. jejuni did not have a significant effect on growth, but the mutation reduced formate respiration and the viability of cells cultured in 5% oxygen. Since cyanide resistance of respiration was diminished in the mutant, we propose that C. jejuni CydAB be renamed CioAB (cyanide-insensitive oxidase), as in Pseudomonas aeruginosa. We measured the oxygen affinity of each oxidase, using a highly sensitive assay that exploits globin deoxygenation during respiration-catalyzed oxygen uptake. The CioAB-type oxidase exhibited a relatively low affinity for oxygen (K(m) = 0.8 microM) and a V(max) of >20 nmol/mg/s. Expression of cioAB was elevated fivefold in cells grown at higher rates of oxygen provision. The alternative, ccoNOQP-encoded cyanide-sensitive oxidase, expected to encode a cytochrome cb'-type enzyme, plays a major role in the microaerobic respiration of C. jejuni, since it appeared to be essential for viability and exhibited a much higher oxygen affinity, with a K(m) value of 40 nM and a V(max) of 6 to 9 nmol/mg/s. Low-temperature photodissociation spectrophotometry revealed that neither oxidase has ligand-binding activity typical of the heme-copper oxidase family. These data are consistent with cytochrome oxidation during photolysis at low temperatures.

How might you compare mitochondria from different tissues and different species?

Mitochondria were isolated from the liver, kidney and mixed hindlimb skeletal muscle of three vertebrate species; the laboratory rat Rattus norvegicus, the bearded dragon lizard Pogona vitticeps, and the cane toad Bufo marinus. These vertebrate species are approximately the same body mass and have similar body temperatures. The content of cytochromes B, C, C1, and A were measured in these isolated mitochondria by oxidised-reduced difference spectra. Adenine nucleotide translocase (ANT) was measured by titration of mitochondrial respiration with carboxyactractyloside and the protein and phospholipid content of isolated mitochondria were also measured. Fatty acid composition of mitochondrial phospholipids was measured. Mitochondrial respiration was measured at 37 degrees C under states III and IV conditions as well as during oligomycin inhibition. Species differed in the ratios of different mitochondrial cytochromes. Muscle mitochondria differed from kidney and liver mitochondria by having a higher ANT content relative to cytochrome content. Respiration rates were compared relative to a number of denominators and found to be most variable when expressed relative to mitochondrial protein content and least variable when expressed relative to mitochondrial cytochrome A and ANT content. The turnover of cytochromes was calculated and found to vary between 1 and 94 electrons s(-1). The molecular activity of mitochondrial cytochromes was found to be significantly positively correlated with the relative polyunsaturation of mitochondrial membrane lipids.

The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins.

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination. METHODS AND RESULTS: In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled. The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes. Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA. The identity of this band was confirmed by its absence in an OmcA-minus mutant. A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method. CONCLUSIONS: Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed. SIGNIFICANCE: The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM. The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g. manganese oxides) and is consistent with its in vivo role.