Customized exogenous ferredoxin functions as an efficient electron carrier
Ontology highlight
ABSTRACT:
SUBMITTER: Song Z
PROVIDER: S-EPMC10992505 | biostudies-literature | 2021 Nov
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.
| S-EPMC7615787 | biostudies-literature
Project description:NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn) is an electron-bifurcating enzyme first discovered in the strict anaerobes Clostridium kluyveri and Moorella thermoacetica. In vivo, Nfn catalyzes the endergonic reduction of NADP+ with NADH coupled to the exergonic reduction of NADP+ with reduced ferredoxin. Most Nfn homologs consist of two subunits, although in certain species Nfn homologs are fused. In contrast to other electron-bifurcating enzymes, Nfn possess a simpler structure. Therefore, Nfn becomes a perfect model to determine the mechanism of flavin-based electron bifurcation, which is a novel energy coupling mode distributed among anaerobic bacteria and archaea. The crystal structures of Nfn from Thermotoga maritima and Pyrococcus furiosus are known, and studies have shown that the FAD molecule of the NfnB (b-FAD) is the site of electron bifurcation, and other cofactors, including a [2Fe2S] cluster, two [4Fe4S] clusters, and the FAD molecule on the NfnA subunit, contribute to electron transfer. Further, the short-lived anionic flavin semiquinone (ASQ) state of b-FAD is essential for electron bifurcation. Nfn homologs are widely distributed among microbes, including bacteria, archaea, and probably eukaryotes, most of which are anaerobes despite that certain species are facultative microbes and even aerobes. Moreover, potential evidence shows that lateral gene transfer may occur in the evolution of this enzyme. Nfn homologs present four different structural patterns, including the well-characterized NfnAB and three different kinds of fused Nfn homologs whose detailed properties have not been characterized. These findings indicate that gene fusion/fission and gene rearrangement may contribute to the evolution of this enzyme. Under physiological conditions, Nfn catalyzes the reduction of NADP+ with NADH and reduced ferredoxin, which is then used in certain NADPH-dependent reactions. Deletion of nfn in several microbes causes low growth and redox unbalance and may influence the distribution of fermentation products. It's also noteworthy that different Nfn homologs perform different functions according to its circumstance. Physiological functions of Nfn indicate that it can be a potential tool in the metabolic engineering of industrial microorganisms, which can regulate the redox potential in vivo.
| S-EPMC6405883 | biostudies-literature
Project description:Fission and fusion can be used to generate new regulatory functions in proteins. This approach has been used to create ferredoxins (Fd) whose cellular electron transfer is dependent upon small molecule binding. To investigate whether Fd fragments can be used to monitor macromolecular binding reactions, we investigated the effects of fusing fragments of Mastigocladus laminosus Fd to single domain antibodies, also known as nanobodies, and their protein antigens. When Fd fragments arising from fission were fused to green fluorescent protein (GFP) and three different anti-GFP nanobodies, split proteins were identified that supported Fd-mediated electron transfer from Fd-NADP reductase (FNR) to sulfite reductase (SIR) in Escherichia coli. However, the order of nanobody and antigen fusion to the Fd fragments affected cellular electron transfer. Insertion of these anti-GFP nanobodies within Fd had differing effects on electron transfer. One domain-insertion variant was unable to support cellular electron transfer unless it was coexpressed with GFP, while others supported electron transfer in the absence of GFP. These findings show how Fds can be engineered so that their electron transfer is regulated by macromolecules, and they reveal the importance of exploring different nanobody homologs and fusion strategies when engineering biomolecular switches.
| S-EPMC11886610 | biostudies-literature
Project description:Proteins from the ferredoxin (Fd) and flavodoxin (Fld) families function as low potential electrical transfer hubs in cells, at times mediating electron transfer between overlapping sets of oxidoreductases. To better understand protein electron carrier (PEC) use across the domains of life, we evaluated the distribution of genes encoding [4Fe-4S] Fd, [2Fe-2S] Fd, and Fld electron carriers in over 7,000 organisms. Our analysis targeted genes encoding small PEC genes encoding proteins having ≤200 residues. We find that the average number of small PEC genes per Archaea (~13), Bacteria (~8), and Eukarya (~3) genome varies, with some organisms containing as many as 54 total PEC genes. Organisms fall into three groups, including those lacking genes encoding low potential PECs (3%), specialists with a single PEC gene type (20%), and generalists that utilize multiple PEC types (77%). Mapping PEC gene usage onto an evolutionary tree highlights the prevalence of [4Fe-4S] Fds in ancient organisms that are deeply rooted, the expansion of [2Fe-2S] Fds with the advent of photosynthesis and a concomitant decrease in [4Fe-4S] Fds, and the expansion of Flds in organisms that inhabit low-iron host environments. Surprisingly, [4Fe-4S] Fds present a similar abundance in aerobes as [2Fe-2S] Fds. This bioinformatic study highlights understudied PECs whose structure, stability, and partner specificity should be further characterized.
| S-EPMC7039249 | biostudies-literature
Project description:PurposeTo better meet clinical needs and facilitate optimal treatment planning, we added two new electron energy beams (7 and 11 MeV) to two Varian TrueBeam linacs.MethodsWe worked with the vendor to create two additional customized electron energies without hardware modifications. For each beam, we set the bending magnet current and then optimized other beam-specific parameters to achieve depths of 50% ionization (I50 ) of 2.9 cm for 7 MeV and 4.2 cm for the 11 MeV beam with the 15 × 15 cm2 cone at 100 cm source-to-surface distance (SSD) by using an ionization chamber profiler (ICP) with a double-wedge (DW) phantom. Beams were steered and balanced to optimize symmetry with the ICP. After all parameters were set, full commissioning was done including measuring beam profiles, percent depth doses (PDDs), output factors (OFs) at standard, and extended SSDs. Measured data were compared between the two linacs and against the values calculated by our RayStation treatment planning system (TPS) following Medical Physics Practice Guideline 5.a (MPPG 5.a) guidelines.ResultsThe I50 values initially determined with the ICP/DW agreed with those from a PDD-scanned in-water phantom within 0.2 mm for the 7 and 11 MeV on both linacs. Comparison of the beam characteristics from the two linacs indicated that flatness and symmetry agreed within 0.4%, and point-by-point differences in PDD were within 0.01% ± 0.3% for the 7 MeV and 0.01% ± 0.3% for the 11 MeV. The OF ratios between the two linacs were 1.000 ± 0.007 for the 7 MeV and 1.004 ± 0.007 for the 11 MeV. Agreement between TPS-calculated outputs and measurements were -0.1% ± 1.0% for the 7 MeV and 0.2% ± 0.8% for the 11 MeV. All other parameters met the MPPG 5.a's 3%/3-mm criteria.ConclusionWe were able to add two new beam energies with no hardware modifications. Tuning of the new beams was facilitated by the ICP/DW system allowing us to have the procedures done in a few hours and achieve highly consistent results across two linacs. PACS numbers: 87.55.Qr, 87.56.Fc.
| S-EPMC9278672 | biostudies-literature
Project description:The activity of ferredoxin (Fd)-dependent cyclic electron flow (Fd-CEF) around photosystem I (PSI) was determined in intact leaves of Arabidopsis thaliana. The oxidation rate of Fd reduced by PSI (vFd) and photosynthetic linear electron flow activity are simultaneously measured under actinic light illumination. The vFd showed a curved response to the photosynthetic linear electron flow activity. In the lower range of photosynthetic linear flow activity with plastoquinone (PQ) in a highly reduced state, vFd clearly showed a linear relationship with photosynthetic linear electron flow activity. On the other hand, vFd increased sharply when photosynthetic linear electron flow activity became saturated with oxidized PQ as the net CO2 assimilation rate increased. That is, under higher photosynthesis conditions, we observed excess vFd resulting in electron flow over photosynthetic linear electron flow. The situation in which excess vFd was observed was consistent with the previous Fd-CEF model. Thus, excess vFd could be attributed to the in vivo activity of Fd-CEF. Furthermore, the excess vFd was also observed in NAD(P)H dehydrogenase-deficient mutants localized in the thylakoid membrane. The physiological significance of the excessive vFd was discussed.
| S-EPMC10419245 | biostudies-literature
Project description:Pseudomonas putida harbors two ferredoxin-NADP(+) reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k(cat)/K(m) value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.
| S-EPMC2648195 | biostudies-literature
Project description:Fixing CO2 via photosynthesis requires ATP and NADPH, which can be generated through linear electron transfer (LET). However, depending on the environmental conditions, additional ATP may be required to fix CO2, which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is determined remains largely unknown. Ferredoxin-NADP+ reductase (FNR) may act as the switch between LET and CET, channeling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or to CET when it is bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. Nevertheless, we circumvented this problem using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing in Chlamydomonas reinhardtii. Through this approach, we created a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth and LET along with enhanced PSI acceptor side limitation relative to the wild type due to slower NADPH reduction. However, the chimeric FNR mutants also showed enhanced ΔpH production and NPQ resulting from increased CET. Overall, our results suggest that rather than promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.
| S-EPMC11912148 | biostudies-literature
Project description:Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy.
| S-EPMC4229662 | biostudies-literature
Project description:The molecular entity responsible for catalyzing ferredoxin (Fd)-dependent cyclic electron flow around photosystem I (Fd-CEF) remains unidentified. To reveal the in vivo molecular mechanism of Fd-CEF, evaluating ferredoxin reduction-oxidation kinetics proves to be a reliable indicator of Fd-CEF activity. Recent research has demonstrated that the expression of Fd-CEF activity is contingent upon the oxidation of plastoquinone. Moreover, chloroplast NAD(P)H dehydrogenase does not catalyze Fd-CEF in Arabidopsis thaliana. In this study, we analyzed the impact of reduced Fd on Fd-CEF activity by comparing wild-type and pgr5-deficient mutants (pgr5hope1). PGR5 has been proposed as the mediator of Fd-CEF, and pgr5hope1 exhibited a comparable CO2 assimilation rate and the same reduction-oxidation level of PQ as the wild type. However, P700 oxidation was suppressed with highly reduced Fd in pgr5hope1, unlike in the wild type. As anticipated, the Fd-CEF activity was enhanced in pgr5hope1 compared to the wild type, and its activity further increased with the oxidation of PQ due to the elevated CO2 assimilation rate. This in vivo research clearly demonstrates that the expression of Fd-CEF activity requires not only reduced Fd but also oxidized PQ. Importantly, PGR5 was found to not catalyze Fd-CEF, challenging previous assumptions about its role in this process.
| S-EPMC10931702 | biostudies-literature