Electron Carrier Molecules Research Articles

Ultrasonication-induced metabolic pathway shifts and reduced electron carrier washout with biomass enhanced hydrogen yield in a continuous stirred tank reactor

Biohydrogen is pivotal for hydrogen economy, providing a renewable, carbon–neutral source of hydrogen from organic waste and biomass. It addresses environmental concerns and supports circular economy principles. However, challenges like managing metabolic pathways and dissolved hydrogen hinder attainment of higher hydrogen yields. This study explored the impact of organic loading rates (OLR) and ultrasonication on glucose fermentation by Clostridium pasteurianum for biohydrogen production.Results showed ultrasonication, particularly at 10 g/L.d OLR, increased hydrogen yield from 1.04 to 1.28 mol-H₂/mol-glucose. Higher OLR boosted biogas production, and at 10 g/L.d OLR, ultrasonication improved hydrogen purity from 46% to 53%, enhancing yield by 13%. C. pasteurianum shifted metabolic pathways under higher OLR, increasing lactate production to address redox imbalance. However, ultrasonication negatively affected lactate and acetate reutilisation, redirecting carbon flux from lactate to butyrate, increasing hydrogen yield.Combining ultrasonication and increased OLR enhanced glycolysis carbon flux by 14.5% and reduced biomass carbon flux by 10.5%. Hydrogen yield, influenced by glycolysis carbon flux, reached 82% under ultrasonication and increased OLR. Ultrasonication mitigated inhibitory effects on electron carrier molecule re-oxidation, emphasising the need to balance biomass, dissolved hydrogen concentration, and OLR for optimal hydrogen recovery.Despite decreased acetate/butyrate ratio and unchanged theoretical hydrogen production with ultrasonication, hydrogen yield increased. This likely resulted from reduced electron carrier molecule wastage with biomass, influenced by higher biomass concentration and ultrasonication. The study underscored preventing electron carrier molecule washout for higher hydrogen production and recommends using batch reactors over continuous stirred tank reactors in dark fermentation operations.

A site-differentiated [4Fe-4S] cluster controls electron transfer reactivity of Clostridium acetobutylicum [FeFe]-hydrogenase I.

One of the many functions of reduction–oxidation (redox) cofactors is to mediate electron transfer in biological enzymes catalyzing redox-based chemical transformation reactions. There are numerous examples of enzymes that utilize redox cofactors to form electron transfer relays to connect catalytic sites to external electron donors and acceptors. The compositions of relays are diverse and tune transfer thermodynamics and kinetics towards the chemical reactivity of the enzyme. Diversity in relay design is exemplified among different members of hydrogenases, enzymes which catalyze reversible H2 activation, which also couple to diverse types of donor and acceptor molecules. The [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI) is a member of a large family of structurally related enzymes where interfacial electron transfer is mediated by a terminal, non-canonical, His-coordinated, [4Fe–4S] cluster. The function of His coordination was examined by comparing the biophysical properties and reactivity to a Cys substituted variant of CaI. This demonstrated that His coordination strongly affected the distal [4Fe–4S] cluster spin state, spin pairing, and spatial orientations of molecular orbitals, with a minor effect on reduction potential. The deviations in these properties by substituting His for Cys in CaI, correlated with pronounced changes in electron transfer and reactivity with the native electron donor–acceptor ferredoxin. The results demonstrate that differential coordination of the surface localized [4Fe–4S]His cluster in CaI is utilized to control intermolecular and intramolecular electron transfer where His coordination creates a physical and electronic environment that enables facile electron exchange between electron carrier molecules and the iron–sulfur cluster relay for coupling to reversible H2 activation at the catalytic site.

Effective Artificial Co-enzyme Based on Single-Electron Reduced Form of 2,2′-Bipyridinium Salt Derivatives for Formate Dehydrogenase in the Catalytic Conversion of CO2 to Formic Acid

Abstract Formate dehydrogenase (FDH) is a useful biocatalyst for CO2 reduction to formic acid in a photoredox system consisting of a photosensitizer and an electron carrier. The electron carrier, single-electron reduced 2,2′-bipyridinium salts (2,2′-BP2+s) act as the co-enzyme for FDH in the reaction of CO2 to formic acid. An advantage of 2,2′-BP2+s is the easy change of structural geometry and the various single-electron reduction potentials. For further improvement of CO2 reduction catalytic activity of FDH, various 2,2′-BP2+s were synthesized as effective artificial co-enzymes for FDH. The effect of the structural geometry and the single-electron reduction potential in the single-electron reduced form of 2,2′-BP2+s on the CO2 reduction catalytic activity of FDH was studied by enzymatic kinetic analysis in detail for the first time. Especially, the catalytic efficiency, kcat/Km value of the single-electron reduced 1,1′-ethylene-2,2′-bipyridinium salt was c.a. 126 times larger than that of native co-enzyme, NADH. These results showed that catalytic activity of FDH can be manipulated with complete control by using 2,2′-BP2+ without changing the structure of FDH and has opened a new avenue for the approach of NAD+/NADH redox free system with FDH using an inexpensive small electron carrier molecule.

A Distinct Aromatic Prenyltransferase Associated with the Futalosine Pathway

Menaquinone (MK) is an electron carrier molecule essential for respiration in most Gram positive bacteria. A crucial step in MK biosynthesis involves the prenylation of an aromatic molecule, catalyzed by integral membrane prenyltransferases of the UbiA (4-hydroxybenzoate oligoprenyltransferase) superfamily. In the classical MK biosynthetic pathway, the prenyltransferase responsible is MenA (1,4-dihydroxy-2-naphthoate octaprenyltransferase). Recently, an alternative pathway for formation of MK, the so-called futalosine pathway, has been described in certain micro-organisms. Until now, five soluble enzymes (MqnA-MqnE) have been identified in the first steps. In this study, the genes annotated as ubiA from T. thermophilus and S. lividans were cloned, expressed and investigated for prenylation activity. The integral membrane proteins possess neither UbiA nor MenA activity and represent a distinct class of prenyltransferases associated with the futalosine pathway that we term MqnP. We identify a critical residue within a highly conserved Asp-rich motif that serves to distinguish between members of the UbiA superfamily.

A novel electron carrier molecule based on a viologen derivative for visible light-driven CO2 reduction to formic acid with the system of zinc porphyrin and formate dehydrogenase

An effective visible light-driven CO2 reduction to formic acid system was developed with zinc porphyrin, formate dehydrogenase and 1,1′-diaminoethyl-4,4′-bipyridinium salt.

Effect of chemical structure of bipyridinium salts as electron carrier on the visible-light induced conversion of CO2 to formic acid with the system consisting of water-soluble zinc porphyrin and formate dehydrogenase

Effect of chemical structures of some 2,2′-bipyridinium salts (BP2+) as the electron carrier molecules on the visible-light induced conversion of CO2 to formic acid with the system consisting of water-soluble zinc tetraphneylporphyrin tetrasulfonate (ZnTPPS) and formate dehydrogenase (FDH) in the presence of triethanolamine (TEOA) as an electron donor molecule was investigated. Irradiation of a CO2 saturated solution containing TEOA, ZnTPPS, BP2+ and FDH with visible light resulted in production of formic acid. By using 1,1′-ethylene-2,2′-bipyridinium dibromide (DB2+) as an electron carrier molecule, the effective formic acid production was observed compared with the other 2,2′-bipyridinium salt derivatives.

Bioinformatic evidence for a widely distributed, ribosomally produced electron carrier precursor, its maturation proteins, and its nicotinoprotein redox partners

BackgroundEnzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses.ResultsAn uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such as N,N-dimethyl-4-nitrosoaniline (NDMA) for the enzyme to cycle.ConclusionsTaken together, these findings suggest that the mycofactocin precursor is modified by the Rv0693 family rSAM protein and other enzymes in its cluster. It becomes an electron carrier molecule that serves in vivo as NDMA and other artificial electron acceptors do in vitro. Subclasses from three different nicotinoprotein families show "only-if" relationships to mycofactocin because they require its presence. This framework suggests a segregated redox pool in which mycofactocin mediates communication among enzymes with non-exchangeable cofactors.

Electrophysiology of Functional Coupling of Electron Transport Chain Complexes

Electron transport chain in the inner mitochondrial membranes consists of four multisubunit protein complexes CI to CIV which are coupled via electron carrier molecules as well as protein-protein interactions. The coupling of the complexes is essential for the proper functioning of the chain and may be an important factor for regulation and balancing of respiration, ATP synthesis and production of reactive oxygen species. However, direct functional studies on the action of the respiratory chain in native surroundings are limited due to the poor accessibility via standard electrophysiological equipment. We performed electrophysiological analyses of electron transport chain in native inner mitochondrial membranes using the solid-supported membranes (SSM) and the SURFE2R technology. The inner mitochondrial membranes were purified from pig heart mitochondria by sucrose gradient fractionation and adsorbed onto SSM sensors. The chain complexes were activated either by NADH for the studies of CI-CIII-CIV coupling, or by succinate and cytochrome c for the analysis of the CII-CIII function. The tested proteins were pharmacologically characterized using specific substrates and inhibitors. Serial application of different inhibitors as well as the coenzyme Q analogs decylubiquinone revealed a tight functional interplay between the complexes CI, CIII, and functional coupling to the complex CIV. The complexes CII and CIII were also functionally coupled. An excess of the coenzyme Q analog idebenone had stimulating effect on the CII-CIII activity but was reducing the CI-CIII-CIV-specific currents. In summary, the presented results demonstrate an easy and reliable approach for studying the complex functional interplay of mitochondrial transport proteins in their native environment, and can help to understand the physiology of different mitochondrial functions. Since different assay conditions can be tested on the same sensor, the technology allows highly effective comparative analysis of the different complex activities.

Soluble Variants of Rhodobacter capsulatus Membrane-anchored Cytochrome cy Are Efficient Photosynthetic Electron Carriers

Photosynthetic (Ps) electron transport pathways often contain multiple electron carriers with overlapping functions. Here we focus on two c-type cytochromes (cyt) in facultative phototrophic bacteria of the Rhodobacter genus: the diffusible cyt c2 and the membrane-anchored cyt c(y). In species like R. capsulatus, cyt c(y) functions in both Ps and respiratory electron transport chains, whereas in other species like R. sphaeroides, it does so only in respiration. The molecular bases of this difference was investigated by producing a soluble variant of cyt c(y) (S-c(y)), by fusing genetically the cyt c2 signal sequence to the cyt c domain of cyt c(y). This novel electron carrier was unable to support the Ps growth of R. capsulatus. However, strains harboring cyt S-c(y) regained Ps growth ability by acquiring mutations in its cyt c domain. They produced cyt S-c(y) variants at amounts comparable with that of cyt c2, and conferred Ps growth. Chemical titration indicated that the redox midpoint potential of cyt S-c(y) was about 340 mV, similar to that of cyts c2 or c(y). Remarkably, electron transfer kinetics from the cyt bc1 complex to the photochemical reaction center (RC) mediated by cyt S-c(y) was distinct from those seen with the cyt c2 or cyt c(y). The kinetics exhibited a pronounced slow phase, suggesting that cyt S-c(y) interacted with the RC less tightly than cyt c2. Comparison of structural models of cyts c2 and S-c(y) revealed that several of the amino acid residues implicated in long-range electrostatic interactions promoting binding of cyt c2 to the RC are not conserved in cyt c(y), whereas those supporting short-range hydrophobic interactions are conserved. These findings indicated that attaching electron carrier cytochromes to the membrane allowed them to weaken their interactions with their partners so that they could accommodate more rapid multiple turnovers.

Different Metabolic Responses in α-, β-, and δ-Cells of the Islet of Langerhans Monitored by Redox Confocal Microscopy

Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic α-, β-, and δ-cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic β-cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non- β-cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified α-, β-, and δ-cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in β- and δ-cell populations in a dose-dependent manner. Conversely, no significant responses were observed in α-cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca 2+ signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the α-, β-, and δ-cell populations, which might be important in the signal transduction events that lead to hormone release.

D-amphetamine-induced depletion of energy and dopamine in the rat striatum is attenuated by nicotinamide pretreatment

The present study examined the effects of nicotinamide on the d-amphetamine (AMPH)-induced dopamine (DA) depletion and energy metabolism change in the rat striatum. In chronic studies, co-administration of AMPH with desipramine, a drug that retards the metabolism of AMPH, (10 mg/kg, intraperitoneal [i.p.], respectively) caused a significant decrease of striatal DA content measured 7 days later. Pretreatment with nicotinamide (500 mg/kg, i.p.), the precursor molecule for the electron carrier molecule nicotinamide adenine dinucleotide (NAD), attenuated this effect of AMPH, whereas itself exerted no long-term effect on striatal DA content. In acute studies, a decrease in striatal adenosine triphospate/adenosine diphosphate (ATP/ADP) ratio was found 3 h after co-injection of AMPH and desipramine. However, nicotinamide pretreatment blocked the reduced striatal ATP/ADP ratio and resulted in a striking increase in striatal NAD content in AMPH-treated rats. Furthermore, nicotinamide was noted to increase striatal ATP/ADP ratio and NAD content in saline-treated rats. These findings suggest that nicotinamide protects against AMPH-induced DAergic neurotoxicity in the striatum of rats via energy supplement.

Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport

The binding of radioactively labelled atrazin, metribuzin and phenmedipham by broken chloroplasts was studied. From the double-reciprocal plots (bound vs. free inhibitors) a high affinity binding reaction is graphically isolated which is related to the inhibition of photosynthetic electron transport. It is concluded that the specific binding sites correspond to the electron carrier molecules which are attacked by the inhibitors. The relative concentration of specific binding sites is 1 per 300–500 chlorophyll molecules. The binding of the labelled substances is competitively inhibited by each of the indicated unlabelled substances, by DCMU and by several pyridazinone derivatives. These results suggest that triazines, triazinones, pyridazinones, biscarbamates and phenylureas interfere with the same electron carrier of the photosynthetic electron transport chain, according to the same molecular mechanism.

Water and cytochrome oxidation-reduction reactions

Heavy water ( 2H 2O) substitution for H 2O in photosynthetic bacterial whole cells and mitochondrial membrane fragments results in an isotope effect of √2 for the ratio of the half-times [ t 1 2 ( 2 H 2 O) /t 1 2 ( H 2 O) ] of oxidation-reduction reactions involving cytochromes. This result, along with the absolute requirement for water, and the effects on the half-times of the substitution of hydrogen-bonding molecules such as ethylene glycol and glycerol for water suggest the direct participation of hydrogen atoms of water and water rotation in the rate-determining step of these electron transfer reactions. Arrhenius plots of cytochrome photooxidation half-times measured at various temperatures from 300 to 77 °K show the half-times to be temperature-dependent from 300 °K to about 150 °K and temperature-independent from about 150 °K through liquid N 2 temperatures. 2H 2O substitution does not change the qualitative response of half-times to temperature; however, the half-times are retarded by √2 throughout the entire temperature range investigated. If the √2 factor found in these experiments is indicative of proton or hydrogen atom motion, then direct electron transfer by electron tunnelling can not be the rate-limiting step in the cytochrome oxidation-reduction processes studied. The experimental results suggest that electron transfer between membrane-bound electron carrier molecules may be effected by means of hydrogen atom transfer via “water bridges” in biological oxidation-reduction reactions.

A new model for mitochondrial membranes based on structural and on biochemical information

On the basis of recent electron microscopic observations by Sjostrand and Barajas23 and available biochemical information, a model describing the molecular structure of mitochondrial membranes was designed. In addition to accounting for the structural features of those membranes as revealed by electron microscopy, this model also pays due attention to the fact that a large number of enzymes and electron carrier molecules are basic components of the membranes. The fundamental justification for such a model which is to assist in an understanding of membrane function is satisfied as far as general principles are concerned. The other justification of such a model concept is to allow predictions regarding additional properties of the membrane. Such predictions have made it possible to design future experiments which can add further information regarding the molecular structure of cellular membranes.