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Nivasorexant, a selective orexin‐1‐receptor antagonist, has recently been assessed in the treatment of humans with binge‐eating disorder. Herein, the inhibitory potential of nivasorexant on cytochromes P450 (CYPs) 2C9, 2C19, and 3A4 was evaluated. Human liver microsomes/recombinant CYP enzymes were evaluated in vitro. In vivo, a single‐center, open‐label, fixed‐sequence study was performed in healthy adults to explore the effect of 100 mg nivasorexant administered twice daily (b.i.d.) on the pharmacokinetics (PK) of flurbiprofen (50 mg, CYP2C9), omeprazole (20 mg, CYP2C19), midazolam (2 mg, CYP3A4) making use of a cocktail approach. Plasma PK sampling was performed over 24 h on Day 1 (Cocktail alone), 8 (Cocktail + nivasorexant), and 15 (Cocktail + nivasorexant at steady state). Genotyping of subjects' CYPs was performed while safety and tolerability were also assessed. In vitro, nivasorexant inhibited CYP2C9, 2C19, and 3A4 in competitive inhibition assays with IC50 values of 8.6, 1.6, and 19–44 μM, respectively, while showing a significant time‐dependent CYP2C19 inhibition. In 22 subjects, exposure to flurbiprofen, omeprazole, and midazolam was generally higher during concomitant single‐ (i.e., area under the plasma concentration–time curve [AUC] ratio increased by 1.04‐, 2.05‐, and 1.56‐fold, respectively) and repeated‐dose (i.e., AUC ratio increased by 1.47‐, 6.84‐, and 3.71‐fold, respectively) nivasorexant administration compared with the cocktail substrates administered alone. The most frequently reported adverse event was somnolence. According to regulatory guidance, nivasorexant is classified as a moderate CYP2C19 and weak CYP3A4 inhibitor after 1 day and as a weak CYP2C9, strong CYP2C19, and moderate CYP3A4 inhibitor after 8 days of 100 mg b.i.d. administration. Clinicaltrials.gov ID: NCT05254548. [ABSTRACT FROM AUTHOR]

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments. [ABSTRACT FROM AUTHOR]

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains. [ABSTRACT FROM AUTHOR]

Background: Neonatal opioid withdrawal syndrome (NOWS), arises due to increased opioid use during pregnancy. Cytochrome P450 (CYP) enzymes play a pivotal role in metabolizing a wide range of substances in the human body, including opioids, other drugs, toxins, and endogenous compounds. The association between CYP gene methylation and opioid effects is unexplored and it could offer promising insights.Objective: To investigate the impact of prenatal opioid exposure on disrupted CYPs in infants and their anticipated long-term clinical implications.Study Design: DNA methylation levels of CYP genes were analyzed in a cohort of 96 placental tissues using Illumina Infinium MethylationEPIC (850 k) BeadChips. This involved three groups of placental tissues: 32 from mothers with infants exposed to opioids prenatally requiring pharmacologic treatment for NOWS, 32 from mothers with prenatally opioid-exposed infants not needing NOWS treatment, and 32 from unexposed control mothers.Results: The study identified 20 significantly differentially methylated CpG sites associated with 17 distinct CYP genes, with 14 CpGs showing reduced methylation across 14 genes (CYP19A1, CYP1A2, CYP4V2, CYP1B1, CYP24A1, CYP26B1, CYP26C1, CYP2C18, CYP2C9, CYP2U1, CYP39A1, CYP2R1, CYP4Z1, CYP2D7P1 and), while 8 exhibited hypermethylation (CYP51A1, CYP26B1, CYP2R1, CYP2U1, CYP4X1, CYP1A2, CYP2W1, and CYP4V2). Genes such as CYP1A2, CYP26B1, CYP2R1, CYP2U1, and CYP4V2 exhibited both increased and decreased methylation. These genes are crucial for metabolizing eicosanoids, fatty acids, drugs, and diverse substances.Conclusion: The study identified profound methylation changes in multiple CYP genes in the placental tissues relevant to NOWS. This suggests that disruption of DNA methylation patterns in CYP transcripts might play a role in NOWS and may serve as valuable biomarkers, suggesting a future pathway for personalized treatment. Further research is needed to confirm these findings and explore their potential for diagnosis and treatment.

Light-induced microbial electron transfer has potential for efficient production of value-added chemicals, biofuels and biodegradable materials owing to diversified metabolic pathways. However, most microbes lack photoactive proteins and require synthetic photosensitizers that suffer from photocorrosion, photodegradation, cytotoxicity, and generation of photoexcited radicals that are harmful to cells, thus severely limiting the catalytic performance. Therefore, there is a pressing need for biocompatible photoconductive materials for efficient electronic interface between microbes and electrodes. Here we show that living biofilms of Geobacter sulfurreducens use nanowires of cytochrome OmcS as intrinsic photoconductors. Photoconductive atomic force microscopy shows up to 100-fold increase in photocurrent in purified individual nanowires. Photocurrents respond rapidly (

Net-casting spiders (Deinopidae) comprise a charismatic family with an enigmatic evolutionary history. There are 67 described species of deinopids, placed among three genera, Deinopis, Menneus, and Asianopis, that are distributed globally throughout the tropics and subtropics. Deinopis and Asianopis, the ogre-faced spiders, are best known for their giant light-capturing posterior median eyes (PME), whereas Menneus does not have enlarged PMEs. Molecular phylogenetic studies have revealed discordance between morphology and molecular data. We employed a character-rich ultra-conserved element (UCE) dataset and a taxon-rich cytochrome-oxidase I (COI) dataset to reconstruct a genus-level phylogeny of Deinopidae, aiming to investigate the group's historical biogeography, and examine PME size evolution. Although the phylogenetic results support the monophyly of Menneus and the single reduction of PME size in deinopids, these data also show that Deinopis is not monophyletic. Consequently, we formally transfer 24 Deinopis species to Asianopis; the transfers comprise all of the African, Australian, South Pacific, and a subset of Central American and Mexican species. Following the divergence of Eastern and Western deinopids in the Cretaceous, Deinopis/Asianopis dispersed from Africa, through Asia and into Australia with its biogeographic history reflecting separation of Western Gondwana as well as long-distance dispersal events.

BackgroundHomalodisca vitripennis Germar, the glassy-winged sharpshooter, is an invasive insect in California and a critical threat to agriculture through its transmission of the plant pathogen, Xylella fastidiosa. Quarantine, broad-spectrum insecticides, and biological control have been used for population management of H. vitripennis since its invasion and subsequent proliferation throughout California. Recently wide-spread neonicotinoid resistance has been detected in populations of H. vitripennis in the southern portions of California's Central Valley. In order to better understand potential mechanisms of H. vitripennis neonicotinoid resistance, we performed RNA sequencing on wild-caught insecticide-resistant and relatively susceptible sharpshooters to profile their transcriptome and population structure.ResultsWe identified 81 differentially expressed genes with higher expression in resistant individuals. The significant largest differentially expressed candidate gene linked to resistance status was a cytochrome P450 gene with similarity to CYP6A9. Furthermore, we observed an over-enrichment of GO terms representing functions supportive of roles in resistance mechanisms (cytochrome P450s, M13 peptidases, and cuticle structural proteins). Finally, we saw no evidence of broad-scale population structure, perhaps due to H. vitripennis' relatively recent introduction to California or due to the relatively small geographic scale investigated here.ConclusionsIn this work, we characterized the transcriptome of insecticide-resistant and susceptible H. vitripennis and identified candidate genes that may be involved in resistance mechanisms for this species. Future work should seek to build on the transcriptome profiling performed here to confirm the role of the identified genes, particularly the cytochrome P450, in resistance in H. vitripennis. We hope this work helps aid future population management strategies for this and other species with growing insecticide resistance.

I present here a personal perspective of the evolution of the two-light reaction two-pigment scheme for the electron transport in oxygenic photosynthesis - as I have lived through it - first as a graduate student of Robert Emerson, from September 1956-January 1959, and then of Eugene Rabinowitch from February 1959-September 1960. I have provided here some of the key published work in my way and have also provided a few photographs. It is essential to remind ourselves upfront that different individuals may have different recollections of the same event (see e.g., the Japanese movie 'Rashomon' https://en.wikipedia.org/wiki/Rashomon). Thus, I encourage others to write about their perspective. Further, I recognize, upfront, the support and encouragement I have received from Robert Blankenship for writing this perspective, and from Győző Garab for submission of this story to Photosynthetica. I begin my perspective with a dedication to Robert Emerson, whose pioneering work led to the concept of the two-light reaction two-pigment system in oxygenic photosynthesis.

ABSTRACTHeme trafficking is a fundamental biological process, yet its direct study has been hampered due to heme’s tight intracellular regulation, heme cytotoxicity, and the transient nature of trafficking. The bacterial System I and System II cytochrome c biogenesis pathways are developing into models to interrogate heme trafficking mechanisms, as they function to transport heme from inside to outside the cell for attachment to apocytochrome c. Cytochromes c require heme for folding and to function in the context of electron transport chains for critical cellular functions, such as respiration. We focus on System I, comprised of eight membrane proteins, CcmABCDEFGH, proposed to function in two steps: CcmABCD mediates the transfer of heme and attachment to CcmE. HoloCcmE chaperones heme to CcmFH for attachment to apocytochrome c. While CcmFH is known to be the holocytochrome c synthase, the mechanism of heme interaction and positioning for attachment to apocytochrome c remains to be elucidated. A comprehensive structure-function analysis of the conserved WWD domain in CcmF was undertaken utilizing alanine-scanning and cysteine-scanning, revealing residues critical for CcmF’s synthase function and residues required for interaction with the 2- and 4-vinyls of heme. This analysis demonstrates for the first time that the CcmF WWD domain directly interacts with heme and that heme interactions within this domain are required for attachment to apocytochrome c. This in-depth interrogation of heme binding now allows for comparison across cytochrome c biogenesis proteins CcmF, CcmC, and CcsBA, revealing common mechanisms of heme interaction in these heme trafficking pathways.IMPORTANCEHeme is an essential co-factor for proteins involved with critical cellular functions, such as energy production and oxygen transport. Thus, understanding how heme interacts with proteins and is moved through cells is a fundamental biological question. This work studies the System I cytochrome c biogenesis pathway, which in some species (including Escherichia coli) is composed of eight integral membrane or membrane-associated proteins called CcmA-H that are proposed to function in two steps to transport and attach heme to apocytochrome c. Cytochrome c requires this heme attachment to function in electron transport chains to generate cellular energy. A conserved WWD heme-handling domain in CcmFH is analyzed and residues critical for heme interaction and holocytochrome c synthase activity are identified. CcmFH is the third member of the WWD domain-containing heme-handling protein family to undergo a comprehensive structure-function analysis, allowing for comparison of heme interaction across this protein family.

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used. Anaerobic methanotrophic archaea play crucial roles in the methane cycle. Here, Zhang et al. provide experimental evidence supporting that multi-heme cytochromes mediate extracellular electron transfer for the reduction of metals and electrodes in these microorganisms. [ABSTRACT FROM AUTHOR]

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient‐specific treatment strategies. Herein, novel 3D vascularized HCC on chips (HCCoCs), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness, are created. HCC inflammation is controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450‐3A4 (CYP3A4) is investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allows for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). It is shown that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increases vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, it is shown that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy‐associated endothelial porosity increase. The HCCoCs are shown to have utility in uncovering the impact of the tumor microenvironment during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies. [ABSTRACT FROM AUTHOR]

I provide here both my personal and scientific autobiography. After giving a background and summary of most of my research, I present information on my parents, my childhood, schooling, university education, and postdoctoral research, all in Australia. This is followed by a presentation of my life and research in Cambridge, UK and then at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), in Australia, since 1955, where most of my research was done, especially on photosynthesis which included the following areas: Purification of a protochlorophyllide-protein complex; separation of the photochemical systems of photosynthesis; development of photochemical activity in photosynthesis; protein synthesis in plants; comparative photosynthesis of sun and shade plants; role of chlorophyll b in photosynthesis; photochemical properties of C4 plants; molecular interaction of thylakoid membranes; electron transport and ATP formation; and solar energy conversion in photosynthesis. In addition to research on the basics and applications of photosynthesis, I also mention at the end my service as a member of the executive of CSIRO. [ABSTRACT FROM AUTHOR]

Mono- and multiheme cytochromes c are post-translationally matured by the covalent attachment of heme. For this, Escherichia coli employs the most complex type of maturation machineries, the Ccm-system (for cytochrome cmaturation). It consists of two membrane protein complexes, one of which shuttles heme across the membrane to a mobile chaperone that then delivers the cofactor to the second complex, an apoprotein:heme lyase, for covalent attachment. Here we report cryo-electron microscopic structures of the heme translocation complex CcmABCD from E. coli, alone and bound to the heme chaperone CcmE. CcmABCD forms a heterooctameric complex centered around the ABC transporter CcmAB that does not by itself transport heme. Our data suggest that the complex flops a heme group from the inner to the outer leaflet at its CcmBC interfaces, driven by ATP hydrolysis at CcmA. A conserved heme-handling motif (WxWD) at the periplasmic side of CcmC rotates the heme by 90° for covalent attachment to the heme chaperone CcmE that we find interacting exclusively with the CcmB subunit. The covalent linkage of hemes to cytochromes c requires a maturation machinery. Here, the authors provide mechanistic insights into how the heme translocase complex CcmABCD flops a heme group, driven by ATP hydrolysis, and delivers it to the chaperone CcmE. [ABSTRACT FROM AUTHOR]

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer. [ABSTRACT FROM AUTHOR]

Improper drug prescription is a main cause of both drug-related harms (inefficacy and toxicity) and ineffective spending and waste of the healthcare system's resources. Nowadays, strategies to support an improved, informed prescription process may benefit from the adequate use of pharmacogenomic testing. Using next-generation sequencing, we analyzed the genomic profile for three major cytochromes P450 (CYP2C9, CYP2C19, CYP2D6) and studied the frequencies of dysfunctional isozymes (e.g., poor, intermediate, or rapid/ultra-rapid metabolizers) in a cohort of 298 Italian subjects. We found just 14.8% of subjects with a fully normal set of cytochromes, whereas 26.5% of subjects had combined cytochrome dysfunction (more than one isozyme involved). As improper drug prescription is more frequent, and more burdening, in polytreated patients, since drug–drug interactions also cause patient harm, we discuss the potential benefits of a more comprehensive PGX testing approach to support informed drug selection in such patients. [ABSTRACT FROM AUTHOR]

Haloalkaliphilic microorganisms are double extremophiles functioning optimally at high salinity and pH. Their typical habitats are soda lakes, geologically ancient yet widespread ecosystems supposed to harbor relict microbial communities. We compared metabolic features and their determinants in two strains of the natronophilic species Dethiobacter alkaliphilus, the only cultured representative of the class "Dethiobacteria" (Bacillota). The strains of D. alkaliphilus were previously isolated from geographically remote Mongolian and Kenyan soda lakes. The type strain AHT1T was described as a facultative chemolithoautotrophic sulfidogen reducing or disproportionating sulfur or thiosulfate, while strain Z-1002 was isolated as a chemolithoautotrophic iron reducer. Here, we uncovered the iron reducing ability of strain AHT1T and the ability of strain Z-1002 for thiosulfate reduction and anaerobic Fe(II) oxidation. Key catabolic processes sustaining the growth of both D. alkaliphilus strains appeared to fit the geochemical settings of two contrasting natural alkaline environments, sulfur-enriched soda lakes and iron-enriched serpentinites. This hypothesis was supported by a meta-analysis of Dethiobacterial genomes and by the enrichment of a novel phylotype from a subsurface alkaline aquifer under Fe(III)-reducing conditions. Genome analysis revealed multiheme c-type cytochromes to be the most probable determinants of iron and sulfur redox transformations in D. alkaliphilus. Phylogeny reconstruction showed that all the respiratory processes in this organism are likely provided by evolutionarily related early forms of unconventional octaheme tetrathionate and sulfite reductases and their structural analogs, OmhA/OcwA Fe(III)-reductases. Several phylogenetically related determinants of anaerobic Fe(II) oxidation were identified in the Z-1002 genome, and the oxidation process was experimentally demonstrated. Proteomic profiling revealed two distinct sets of multiheme cytochromes upregulated in iron(III)- or thiosulfate-respiring cells and the cytochromes peculiar for Fe(II) oxidizing cells. We suggest that maintaining high variation in multiheme cytochromes is an effective adaptive strategy to occupy geochemically contrasting alkaline environments. We propose that sulfurenriched soda lakes could be secondary habitats for D. alkaliphilus compared to Fe-rich serpentinites, and that the ongoing evolution of Dethiobacterales could retrace the evolutionary path that may have occurred in prokaryotes at a turning point in the biosphere's history, when the intensification of the sulfur cycle outweighed the global significance of the iron cycle. [ABSTRACT FROM AUTHOR]

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.

An important role in ensuring the functioning of the respiratory chain belongs to the cytochrome part, which includes complexes III (ubiquinol-cytochrome c oxidoreductase) and IV (cytochrome c oxidase). The key components of these enzymatic complexes are heme-containing cytochromes, the number of which depends on the balance of heme synthesis and catabolism. δ-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway, while heme oxygenase is the key enzyme of heme degradation. It is known that nutritional imbalances drive many risk factors for chronic kidney disease. That is why our research aimed to study the activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes, the level of cytochromes a+a3, b, c, and c1, and the activity of key enzymes of heme metabolism in the mitochondria of rat kidneys under conditions of different content of protein and sucrose in animal diet. The obtained results showed a decreased activity of ubiquinol-cytochrome c oxidoreductase and cytochrome oxidase complexes and reduced levels of mitochondria cytochromes a+a3, b, c, and c1 in the kidney mitochondria under the conditions of nutrient imbalance, with the most pronounced changes found in animals kept on a low-protein/high-sucrose diet. A decrease in δ-aminolevulinate synthase activity with a simultaneous 2-fold increase in heme oxygenase activity was found in kidney mitochondria of animals kept on a low-protein/high-sucrose diet compared to those kept on full-value diet indicating an intensification of heme catabolism along with inhibition of its synthesis. The obtained results testify the energy imbalance under the conditions of low-protein/high-sucrose which in turn can lead to the progression of kidney injury.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction. [ABSTRACT FROM AUTHOR]

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, andO2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c0 (cyts c0) are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c0-a) and an unrelated family with a large betasheet fold (cyts c0-ß) resembling that of cytochromes P460. A recent structure of cyt c0-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c0-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c0-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins. [ABSTRACT FROM AUTHOR]

In the biosynthesis of the tryptophan‐linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high‐resolution crystal structure of selective Csp2−N bond forming dimerase, AspB. Overlay of the AspB structure onto C−C and C−N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C−N bond formation. MD simulations also suggest that intermolecular C−C or C−N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic. [ABSTRACT FROM AUTHOR]

The recent development of tailored cytochrome enzymes has enabled "new‐to‐nature" reactivities, such as the biocatalytic formation of carbon‐silicon bonds using the cytochrome c from Rhodothermus marinus. To maximise the potential of this remarkable biocatalyst by increasing its turnover numbers (TON) and to enable its reusability in continuous processes, we report the use of the SpyTag/SpyCatcher bioconjugation system to immobilise this enzyme. We successfully modified the enzyme with a SpyTag without significant effects on its catalytic activity. Even after immobilization on microparticles the enzyme retained 60 % activity. When the immobilized enzyme was used in sequential batch or continuous flow to produce an organosilicon, we observed up to 6‐fold higher turnover numbers over a total period of 10 days compared to the free enzyme reaction, however we observed a drop in stereoselectivity under these conditions. This is the first report on the successful immobilisation of a carbon‐silicon bond forming enzyme for the continuous, biocatalytic production of organosilicons. [ABSTRACT FROM AUTHOR]

To study magnetoreception of Arabidopsis thaliana, we analysed several developmental responses including cryptochrome-independent seed germination and the phytochrome- and cryptochrome-dependent hypocotyl elongation and photo-accumulation of anthocyanins and chlorophylls in weak static magnetic fields ranging from near null to 122 μT. A field of 50 μT accelerated seed germination by about 20 h relative to samples maintained in a near-null field. The double mutant, cry1cry2, lacking cryptochromes 1 and 2 displayed the same magnetic field–induced germination acceleration under blue light as the wild-type strain. Magnetic field–induced germination acceleration was masked in the presence of exogenous sucrose. Stimulus–response curves for hypocotyl elongation in a range between near-null to 122 μT indicated maxima near 9 and 60 μT for the wild-type strain as well as mutant cry1cry2. The photo-accumulation of anthocyanins and chlorophylls could be effectively modulated by magnetic fields in the presence of low-irradiance red and blue light, respectively. The findings indicate that Arabidopsis thaliana possesses light-independent mechanisms of magnetic field reception, which remain presently unidentified. Our results are in better agreement with predictions of the level crossing mechanism (LCM) of magnetoreception rather than those of the cryptochrome-associated radical-pair mechanism (RPM). [ABSTRACT FROM AUTHOR]

Heme is an iron-protoporphyrin complex with an essential physiologic function for all cells, especially for those in which heme is a key prosthetic group of proteins such as hemoglobin, myoglobin, and cytochromes of the mitochondria. However, it is also known that heme can participate in pro-oxidant and pro-inflammatory responses, leading to cytotoxicity in various tissues and organs such as the kidney, brain, heart, liver, and in immune cells. Indeed, heme, released as a result of tissue damage, can stimulate local and remote inflammatory reactions. These can initiate innate immune responses that, if left uncontrolled, can compound primary injuries and promote organ failure. In contrast, a cadre of heme receptors are arrayed on the plasma membrane that is designed either for heme import into the cell, or for the purpose of activating specific signaling pathways. Thus, free heme can serve either as a deleterious molecule, or one that can traffic and initiate highly specific cellular responses that are teleologically important for survival. Herein, we review heme metabolism and signaling pathways, including heme synthesis, degradation, and scavenging. We will focus on trauma and inflammatory diseases, including traumatic brain injury, trauma-related sepsis, cancer, and cardiovascular diseases where current work suggests that heme may be most important. [ABSTRACT FROM AUTHOR]

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes. [ABSTRACT FROM AUTHOR]

The development of environment‐friendly, step economic couplings to generate structurally diverse macrocyclic compounds is highly desirable but poses a marked challenge. Inspired by the C−H oxidation mechanism of cytochromes P450, an unprecedented and practical RhIII‐catalyzed acylmethylation macrocyclization via C−H/O2 dual activation has been developed by us. The process of macrocyclization is facilitated by a synergic coordination from pyridine and ester group. Interestingly, the reaction mode derives from a three‐component coupling which differs from established olefination and alkylation paths. Density functional theory (DFT) calculations and control experiments revealed the mechanism of this unique C−H/O2 dual activation. The newly achieved acylmethylation macrocyclic products and their derivatives showed a potent anti‐H1N1 bioactivity, which may provide an opportunity for the discovery of novel anti‐H1N1 macrocyclic leading compounds. [ABSTRACT FROM AUTHOR]

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme–albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins. [ABSTRACT FROM AUTHOR]

The effects of two anticancer active copper(II) mixed-ligand complexes of the type [Cu(qui)(mphen)]Y·H2O, where Hqui = 2-phenyl-3-hydroxy- 1H-quinolin-4-one, mphen = bathophenanthroline, and Y = NO3 (complex 1) or BF4 (complex 2) on the activities of different isoenzymes of cytochrome P450 (CYP) have been evaluated. The screening revealed significant inhibitory effects of the complexes on CYP3A4/5 (IC50 values were 2.46 and 4.88 μM), CYP2C9 (IC50 values were 16.34 and 37.25 μM), and CYP2C19 (IC50 values were 61.21 and 77.07 μM). Further, the analysis of mechanisms of action uncovered a non-competitive type of inhibition for both the studied compounds. Consequent studies of pharmacokinetic properties proved good stability of both the complexes in phosphate buffer saline (>96% stability) and human plasma (>91% stability) after 2 h of incubation. Both compounds are moderately metabolised by human liver microsomes (<30% after 1 h of incubation), and over 90% of the complexes bind to plasma proteins. The obtained results showed the potential of complexes 1 and 2 to interact with major metabolic pathways of drugs and, as a consequence of this finding, their apparent incompatibility in combination therapy with most chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

BackgroundMost species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1.ResultIn the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome.ConclusionOur results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c‐type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA‐rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood‐Ljundahl pathway for acetyl‐CoA synthesis, whereas the ∆mmcA‐rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems‐level insights into the non‐overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA. [ABSTRACT FROM AUTHOR]

Metallic nanoparticles have been extensively used to adsorb biomolecules and improve their stability under broader conditions. This study focuses on the interaction of Cytochrome C (CytC) and gold nanoparticles (AuNP), CytC@AuNP. Circular dichroism (CD) and Raman show significant differences in secondary structure, mainly in α‐helix. CD ramp temperatures revealed enhanced thermal stability of CytC@AuNP, as evidenced by a refolding of the α‐helix conformation of CytC and a delay in denaturation temperature by approximately 15 °C. Catalytic oxidation performed with CytC@AuNP showed a hyperbolic profile up to 104 % higher yields compared to CytC, which exhibited a linear profile and 30 % loss of activity after thermal treatment. The same reaction performed upon plasmon stimulus comparatively increased the catalytic yield of CytC@AuNP in 12 %, although it had no effect on free CytC. This study highlights the advantages of adsorbing biomolecules on nanoparticles and is a glimpse at plasmon‐driven nanobiocatalysis. [ABSTRACT FROM AUTHOR]

Tamalia (Hemiptera: Aphididae: Tamaliinae), a Nearctic aphid genus, is associated with galls on woody plants in the family Ericaceae (Arctostaphylos spp., Arbutus arizonica , and Comarostaphylis diversifolia). Tamalia cruzensis Miller and Pike, n. sp., Tamalia glaucensis Miller and Pike, n. sp., and Tamalia moranae Miller and Pike, n. sp. are described and illustrated. Two of these, T. cruzensis and T. moranae , represent host plant records for Tamalia on genera other than Arctostaphylos spp. Character measurements, comparisons, and descriptions; DNA cytochrome oxidase subunit 1 sequences; geographic distributions; seasonal occurrence; biology; and host plant associations are provided, along with diagnoses and a key to the known species based on the gall-inhabiting apterous adult stage. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) are the most important human drug metabolizing enzymes, but their mutual interactions are poorly understood. In this study, we recombinantly co-expressed of each one of the 19 human members of the UGT families 1 and 2 with either CYP2C9, CYP2D6, or CYP4Z1 in fission yeast. Using these strains, we monitored a total of 72 interactions: 57 cases where we tested the influence of UGT co-expression on CYP activity and 15 cases of the opposite approach. In the majority of cases (88%), UGT co-expression had a statistically significant (p < 0.05) effect on P450 activity (58% positive and 30% negative). Strong changes were observed in nine cases, including one case with an activity increase by a factor of 23 (CYP2C9 activity in the presence of UGT2A3) but also four cases with a complete loss of activity. When monitoring the effect of CYP co-expression on the activity of five UGTs, activity changes were generally not so pronounced and, if observed, always detrimental. UGT2B7 activity was not influenced by CYP co-expression, while the other UGTs were affected to varying degrees. These data suggest the notion that mutual influence of CYPs and UGTs on each other's activity is a widespread phenomenon. [ABSTRACT FROM AUTHOR]

Introduction: Most hepatically cleared drugs are metabolized by cytochromes P450 (CYPs), and Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines provide curated clinical references for CYPs to apply individual genome data for optimized drug therapy. However, incorporating novel pharmacogenetic variants into guidelines takes considerable time. Methods: We comprehensively assessed the drug metabolizing capabilities of CYP2C19 variants discovered through population sequencing of two substrates, S-mephenytoin and omeprazole. Results: Based on established functional assays, 75% (18/24) of the variants not yet described in Pharmacogene Variation (PharmVar) had significantly altered drug metabolizing capabilities. Of them, seven variants with inappreciable protein expression were evaluated as protein damaging by all three in silico prediction algorithms, Sorting intolerant from tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), and Combined annotation dependent depletion (CADD). The five variants with decreased metabolic capability (<50%) of wild type for either substrates were evaluated as protein damaging by all three in silico prediction algorithms, except CADD exact score of NM_000769.4:c.593T>C that was 19.68 (<20.0). In the crystal structure of the five polymorphic proteins, each altered residue of all those proteins was observed to affect the key structures of drug binding specificity. We also identified polymorphic proteins indicating different tendencies of metabolic capability between the two substrates (5/24). Discussion: Therefore, we propose a methodology that combines in silico prediction algorithms and functional assays on polymorphic CYPs with multiple substrates to evaluate the changes in the metabolism of all possible genomic variants in CYP genes. The approach would reinforce existing guidelines and provide information for prescribing appropriate medicines for individual patients. [ABSTRACT FROM AUTHOR]

Cytochromes are enzymes of the dehydrogenase class with a hemoprotein structure in which the iron in these compounds undergoes oxidation and reduction reactions upon receiving or losing electrons. Quantitatively speaking, CYP3A4 is the most important isoenzyme of cytochrome P450, oxidizing foreign organic molecules such as drugs or toxins to cause them to leave the body. Many drugs and antibiotics can induce or inhibit the activity of cytochrome P450, including dexamethasone. Dexamethasone is a steroidal anti-inflammatory drug used to treat of inflammatory diseases and chronic autoimmunity. This study aimed to investigate the induction effect of dexamethasone in biotransformation pathways by in silico tools. Molegro Virtual Docker software was used to investigate the molecular docking of the enzyme and dexamethasone, which indicated the binding of the drug to the enzyme. The molecular simulation was performed in Linux with the GROMACS program. Root-mean-square distance (RMSD), and radius of gyration (Rg), were evaluated. The results were analyzed with Pymol and VMD software, and the obtained curve was plotted with GRACE software. Docking results show that a cluster with a bond energy of -60.81 was the best cluster, and the bond size between ligand and internal atoms was -23.191in the complex. In addition, the amount of bond between the ligand and water for this pose was zero. The stability of the enzyme-ligand complex and the induction effect of dexamethasone on CYP3A4 were indicated by RMSD and RG results. Results of RMSD and RG of CYP3A4 glucocorticoid obtained from the simulation showed the stability of binding of the drug to the enzyme. Also, RMSD results showed the stability of glucocorticoid and dexamethasone complex during molecular dynamics simulation. It reached relative stability at 0.8 nm after 80,000 ps until the end of the simulation. [ABSTRACT FROM AUTHOR]

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated. At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes. Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes. Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2–1.5. These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes. [ABSTRACT FROM AUTHOR]

I present here a personal perspective of the evolution of the two-light reaction two-pigment scheme for the electron transport in oxygenic photosynthesis – as I have lived through it – first as a graduate student of Robert Emerson, from September 1956–January 1959, and then of Eugene Rabinowitch from February 1959–September 1960. I have provided here some of the key published work in my way and have also provided a few photographs. It is essential to remind ourselves upfront that different individuals may have different recollections of the same event (see e.g., the Japanese movie ‘Rashomon’ https://en.wikipedia.org/wiki/Rashomon). Thus, I encourage others to write about their perspective. Further, I recognize, upfront, the support and encouragement I have received from Robert Blankenship for writing this perspective, and from Győző Garab for submission of this story to Photosynthetica. I begin my perspective with a dedication to Robert Emerson, whose pioneering work led to the concept of the two-light reaction two-pigment system in oxygenic photosynthesis. [ABSTRACT FROM AUTHOR]

Long-range (>10 μm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within ∼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.

Electrons can be transported from microbes to external insoluble electron acceptors (e.g., metal oxides or electrodes in an electrochemical cell). This process is known as extracellular electron transfer (EET) and has received considerable attention due to its applications in environmental remediation and energy conversion. However, the paucity of rapid and noninvasive phenotyping techniques hinders a detailed understanding of microbial EET mechanisms. Most EET phenotyping techniques assess microorganisms based on their metabolism and growth in various conditions and/or performance in electrochemical systems, which requires large sample volumes and cumbersome experimentation. Here, we use microfluidic dielectrophoresis to show a strong correlation between bacterial EET and surface polarizability. We analyzed surface polarizabilities for wild-type strains and cytochrome-deletion mutants of two model EET microbes, Geobacter sulfurreducens and Shewanella oneidensis, and for Escherichia coli strains heterologously expressing S. oneidensis EET pathways in various growth conditions. Dielectrophoretic phenotyping is achieved with small cell culture volumes (~100 μl) in a short amount of time (1 to 2 min per strain). Our work demonstrates that cell polarizability is diminished in response to deletions of crucial outer-membrane cytochromes and enhanced due to additions of EET pathways. Results of this work hold exciting promise for rapid screening of direct EET or other cell envelope phenotypes using cell polarizability as a proxy, especially for microbes difficult to cultivate in laboratory conditions.

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of heterologous cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the steroid nucleus enzymatic degradation. Three sets of genetic constructs have been created for heterologous expression of the genes encoding P450 cytochromes CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from B. megaterium ATCC13368 in M. smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (pNS31 and pNS32), tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and adrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15β-hydroxyandrostenedione (15β-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15β-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylation activity. The results we obtained are of a priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2, and are important for the creation of microbial strains for producing valuable hydroxysteroids. [ABSTRACT FROM AUTHOR]

Photosynthesis converts light energy to chemical energy to fuel life on earth. Light energy is harvested by antenna pigments and transferred to reaction centers (RCs) to drive the electron transfer (ET) reactions. Here, we present cryo-electron microscopy (cryo-EM) structures of two forms of the RC from the microaerophilic Chloracidobacterium thermophilum (CabRC): one containing 10 subunits, including two different cytochromes; and the other possessing two additional subunits, PscB and PscZ. The larger form contained 2 Zn-bacteriochlorophylls, 16 bacteriochlorophylls, 10 chlorophylls, 2 lycopenes, 2 hemes, 3 Fe4S4 clusters, 12 lipids, 2 Ca2+ ions and 6 water molecules, revealing a type I RC with an ET chain involving two hemes and a hybrid antenna containing bacteriochlorophylls and chlorophylls. Our results provide a structural basis for understanding the excitation energy and ET within the CabRC and offer evolutionary insights into the origin and adaptation of photosynthetic RCs. During photosynthesis, light energy is harvested by an antenna systems and delivered to the reaction centers (RCs) for charge separation and electron transfer (ET). The authors report cryo-EM structures of two forms of RC from the microaerophilic Chloracidobacterium thermophilum (CabRC), providing a structural basis for ET within the CabRC. [ABSTRACT FROM AUTHOR]

Theodore "Ted" Kuwana and his research group played a central role in the invention and early development of spectroelectrochemistry at optically transparent electrodes (OTEs). The history of the "idea" and the first ten years of research are reviewed. Topics include OTEs and spectroelectrochemical cells for transmission and internal reflection experiments, rapid scan spectrometer development, and applications to kinetics of homogeneous chemical reactions associated with electrode processes and redox active biological compounds such as cytochromes. Relevant work of other contemporary research groups is also presented. [ABSTRACT FROM AUTHOR]

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4‐cyclohexylbenzoic acid but not the aromatic oxidation of 4‐phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4‐benzyl‐, 4‐phenoxy‐ and 4‐benzoyl‐benzoic acid and methoxy‐substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4‐benzylbenzoic acid. The methoxy‐substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4‐(3′‐methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4‐phenylbenzoic acid. 4‐Phenoxy‐ and 4‐benzoyl‐benzoic acid bound with the ether or ketone oxygen atom hydrogen‐bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4‐phenylbenzoic acid; however the F182L mutant was able to catalyse 4‐phenylbenzoic acid oxidation generating 2′‐hydroxy‐, 3′‐hydroxy‐ and 4′‐hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild‐type enzyme, the phenyl ring of 4‐phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur. [ABSTRACT FROM AUTHOR]

Cytochrome P450 is an enzyme involved in the metabolism of phase 1 xenobiotics, toxins, endogenous hormones, and drugs, including those used in COVID-19 treatment. Cytochrome p450 genes are linked to the pathogenesis of some multifactorial traits and diseases, such as cancer, particularly prostate cancer, colorectal cancer, breast cancer, and cervical cancer. Genotyping was performed on 540 supposedly healthy individuals of 5 Finno-Permic populations from the territories of the European part of the Russian Federation. There was a statistically significant difference between Veps and most of the studied populations in the rs4986774 locus of the CYP2D6 gene; data on the rs3892097 locus of the CYP2D6 gene shows that Izhemsky Komis are different from the Mordovian and Udmurt populations. [ABSTRACT FROM AUTHOR]

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin. [ABSTRACT FROM AUTHOR]

We present here a tribute to one of the foremost biophysicists of our time, Vladimir Anatolievich Shuvalov, who made important contributions in bioenergetics, especially on the primary steps of conversion of light energy into charge-separated states in both anoxygenic and oxygenic photosynthesis. For this, he and his research team exploited pico- and femtosecond transient absorption spectroscopy, photodichroism & circular dichroism spectroscopy, light-induced FTIR (Fourier-transform infrared) spectroscopy, and hole-burning spectroscopy. We remember him for his outstanding leadership and for being a wonderful mentor to many scientists in this area. Reminiscences by many [Suleyman Allakhverdiev (Russia); Robert Blankenship (USA); Richard Cogdell (UK); Arvi Freiberg (Estonia); Govindjee Govindjee (USA); Alexander Krasnovsky, jr, (Russia); William Parson (USA); Andrei Razjivin (Russia); Jian- Ren Shen (Japan); Sergei Shuvalov (Russia); Lyudmilla Vasilieva (Russia); and Andrei Yakovlev (Russia)] have included not only his wonderful personal character, but his outstanding scientific research. [ABSTRACT FROM AUTHOR]

Cytochromes c use heme as a cofactor to carry electrons in respiration and photosynthesis. The cytochrome c maturation system I, consisting of eight membrane proteins (CcmABCDEFGH), results in the attachment of heme to cysteine residues of cytochrome c proteins. Since all c-type cytochromes are periplasmic, heme is first transported to a periplasmic heme chaperone, CcmE. A large membrane complex, CcmABCD has been proposed to carry out this transport and linkage to CcmE, yet the structural basis and mechanisms underlying the process are unknown. We describe high resolution cryo-EM structures of CcmABCD in an unbound form, in complex with inhibitor AMP-PNP, and in complex with ATP and heme. We locate the ATP-binding site in CcmA and the heme-binding site in CcmC. Based on our structures combined with functional studies, we propose a hypothetic model of heme trafficking, heme transfer to CcmE, and ATP-dependent release of holoCcmE from CcmABCD. CcmABCD represents an ABC transporter complex using the energy of ATP hydrolysis for the transfer of heme from one binding partner (CcmC) to another (CcmE). Bacterial ABC transporter complex CcmABCD, a part of cytochrome c maturation system, transfers heme from one binding partner (CcmC) to another (CcmE). Here authors describe high resolution cryo-EM structures of CcmABCD in multiple states and propose a hypothetic model of heme trafficking, heme transfer and release of holoCcmE. [ABSTRACT FROM AUTHOR]

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third‐generation sequencing and novel analytical toolsets. Applying these techniques to woodrats (Neotoma spp.) provides a unique opportunity to study how herbivores respond to environmental change. Neotoma bryanti and N. lepida independently achieved a major dietary feat in the aftermath of a natural climate change event: switching to the novel, toxic food source creosote bush (Larrea tridentata). To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti × N. lepida F1 hybrid, allowing the simultaneous assembly of genomes representing each parental species. The resulting phased, chromosome‐level, highly complete haploid references enabled us to explore the genomic architecture of several gene families—cytochromes P450, UDP‐glucuronosyltransferases (UGTs), and ATP‐binding cassette (ABC) transporters—known to play key roles in the metabolism of naturally occurring toxic dietary compounds. In addition to duplication events in the ABCG and UGT2B subfamilies, we found expansions in three P450 gene families (2A, 2B, 3A), including the evolution of multiple novel gene islands within the 2B and 3A subfamilies, which may have provided the crucial substrate for dietary adaptation. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago. [ABSTRACT FROM AUTHOR]