Engineering of Phycourobilin Synthase: PubS to a Two-Electron Reductase.

Phycourobilin:ferredoxin oxidoreductase (PubS) belongs to the ferredoxin-dependent bilin reductase (FDBR) family and catalyzes the reduction of the C15=C16 double bond, followed by the C4=C5 double bond of biliverdin IXα to produce phycourobilin. Among the diverse FDBR enzymes that catalyze site-specific reduction reactions of bilins, PubS lineage is the only one that reduces the C4=C5 double bond. This family can be broadly divided into four-electron reduction enzymes, which catalyze two successive two-electron reductions, such as PubS, and two-electron reduction enzymes, which catalyze a single two-electron reduction. The crystal structures of diverse FDBRs, excluding PubS, have unraveled that there are two distinct binding modes in the substrate-binding pocket. In this study, we focused on the arginine (Arg) residues that is considered crucial for substrate-binding mode in two-electron reduction enzymes. Through sequence alignment and comparison with the predicted structure of PubS, we identified a residue in PubS that corresponds to the Arg residue in the two-electron reduction enzymes. We further introduced mutations to avoid the steric hindrance associated with changes in the binding mode. Biochemical characterization of these variants showed that we successfully modified PubS from a four-electron reduction enzyme to a two-electron reduction enzyme with the accumulation of radicals. Our results provide insight into the molecular mechanisms of the chromophore binding mode and proton donation from acidic residues.

Insights into electron transfer and bifurcation of the Synechocystis sp. PCC6803 hydrogenase reductase module.

The NAD+-reducing soluble [NiFe] hydrogenase (SH) is the key enzyme for production and consumption of molecular hydrogen (H2) in Synechocystis sp. PCC6803. In this study, we focused on the reductase module of the SynSH and investigated the structural and functional aspects of its subunits, particularly the so far elusive role of HoxE. We demonstrated the importance of HoxE for enzyme functionality, suggesting a regulatory role in maintaining enzyme activity and electron supply. Spectroscopic analysis confirmed that HoxE and HoxF each contain one [2Fe2S] cluster with an almost identical electronic structure. Structure predictions, alongside experimental evidence for ferredoxin interactions, revealed a remarkable similarity between SynSH and bifurcating hydrogenases, suggesting a related functional mechanism. Our study unveiled the subunit arrangement and cofactor composition essential for biological electron transfer. These findings enhance our understanding of NAD+-reducing [NiFe] hydrogenases in terms of their physiological function and structural requirements for biotechnologically relevant modifications.

AB017. NFκB1 regulates FDX1 to facilitate elesclomol-induced cuproptosis against glioblastoma.

BACKGROUND: Ferredoxin 1 (FDX1) plays key roles in promoting elesclomol-induced cuproptosis against cancer, whether it has the potential to be a new therapeutic strategy against glioblastoma has not yet been clarified. METHODS: Glioblastoma cells were treated with elesclomol (20 nM/L) and copper chloride (2 µM/L), and then cell proliferation, migration, and invasion were evaluated by CCK-8, clonogenic and transwell assay. Western blot was performed to detect the expression of cuproptosis-relating proteins FDX1, lipoylated dihydrolipoamide S-acetyltransferase (DLAT), copper transport ATPase (ATP7A), heat shock protein 70 (HSP70), apoptotic markers B cell lymphoma-2 (BCL-2) associated X protein (Bax), and BCL-2, as well as the pan-apoptotic/death markers gasdermin D (GSDMD), solute carrier family 7 member 11 (SLC7A11). The effects of knockdown and overexpression of FDX1 on cell proliferation, migration, and invasion were observed. Bioinformatic analysis was performed to predict the corresponding transcription factors regulating FDX1 expression, and verified by dual luciferase assay. The regulatory relationship between FDX1 and its transcription factors was verified by rescue experiment and further evaluated in vivo. RESULTS: Elesclomol had obvious inhibitory effects on the proliferation, migration, and invasion capacities of tumor cells in a dose-dependent manner. When combined with copper chloride, the inhibitory effects on tumor cells were significantly higher both in vitro and in vivo. FDX1 expression was negatively correlated with overall survival of patients. Nuclear factor κ-light-chain enhancer of activated B cell 1 (NFκB1) was the transcription factor of FDX1 verified by dual luciferase assay. Both FDX1 and NFκB1 were highly expressed in glioblastoma. Knockdown of FDX1 or NFκB1 down-regulated proliferation, migration, and invasion abilities of tumor cells, and increased after FDX1 overexpression. FDX1 expression decreased correspondingly after NFκB1 knockdown. Up-regulation of FDX1 promoted elesclomol-induced cuproptosis against glioblastoma both in vitro and in vivo. FDX1 knockdown can reverse the inhibitory effect of elesclomol on tumor cells. CONCLUSIONS: Elesclomol inhibits glioblastoma development via inducing cuproptosis, regulated by NFκB1/FDX1 axis.

The rapid-reaction kinetics of an electron-bifurcating flavoprotein, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase EtfAB:bcd.

We have investigated the kinetic behavior of the electron-bifurcating crotonyl-CoA-dependent NADH: ferredoxin oxidoreductase EtfAB:bcd from Megasphaera elsdenii. The overall behavior of the complex in both the reductive and the oxidative half-reactions is consistent with that previously determined for the individual EtfAB and bcd components. This includes an uncrossing of the half-potentials of the bifurcating flavin of the EtfAB component in the course of ferredoxin-reducing catalysis, ionization of the bcd flavin semiquinone and the appearance of a charge transfer complex upon binding of the high potential acceptor crotonyl-CoA. The observed rapid-reaction rates of ferredoxin reduction are independent of [NADH], [crotonyl-CoA], and [ferredoxin], with an observed rate of ∼0.2 s-1, consistent with the observed steady-state kinetics. In enzyme-monitored turnover experiments, an approach to steady-state where the complex's flavins become reduced but no ferredoxin is generated is followed by a steady-state phase characterized by extensive ferredoxin reduction but little change in overall levels of flavin reduction. The approach to the steady-state phase can be eliminated by prior reduction of the complex, in which case there is no lag in the onset of ferredoxin reduction; this is consistent with the et FAD needing to be reduced to the level of the (anionic) semiquinone for bifurcation and concomitant ferredoxin reduction to occur. Single-turnover experiments support this conclusion, with the accumulation of the anionic semiquinone of the et FAD apparently required to prime the system for subsequent bifurcation and ferredoxin reduction.

Ferredoxins: Functions, Evolution, Potential Applications, and Challenges of Subtype Classification.

Ferredoxins are proteins found in all biological kingdoms and are involved in essential biological processes including photosynthesis, lipid metabolism, and biogeochemical cycles. Ferredoxins are classified into different groups based on the iron-sulfur (Fe-S) clusters that they contain. A new subtype classification and nomenclature system, based on the spacing between amino acids in the Fe-S binding motif, has been proposed in order to better understand ferredoxins' biological diversity and evolutionary linkage across different organisms. This new classification system has revealed an unparalleled diversity between ferredoxins and has helped identify evolutionarily linked ferredoxins between species. The current review provides the latest insights into ferredoxin functions and evolution, and the new subtype classification, outlining their potential biotechnological applications and the future challenges in streamlining the process.

FDX1 downregulation activates mitophagy and the PI3K/AKT signaling pathway to promote hepatocellular carcinoma progression by inducing ROS production.

BACKGROUND: Mitochondrial dysfunction and metabolic reprogramming can lead to the development and progression of hepatocellular carcinoma (HCC). Ferredoxin 1 (FDX1) is a small mitochondrial protein and recent studies have shown that FDX1 plays an important role in tumor cuproptosis, but its role in HCC is still elusive. In this study, we aim to investigate the expression and novel functions of FDX1 in HCC. METHODS: FDX1 expression was first analyzed in publicly available datasets and verified by immunohistochemistry, qRT-PCR and Western blot. In vitro and in vivo experiments were applied to explore the functions of FDX1. Non-targeted metabolomics and RNA-sequencing were used to determine molecular mechanism. mRFP-GFP-LC3 lentivirus transfection, Mito-Tracker Red and Lyso-Tracker Green staining, transmission electron microscopy, flow cytometry, JC-1 staining, etc. were used to analyze mitophagy or ROS levels. Hydrodynamic tail vein injection (HTVi) and patient-derived organoid (PDO) models were used to analyze effect of FDX1 overexpression. RESULTS: FDX1 expression is significantly downregulated in HCC tissues. FDX1 downregulation promotes HCC cell proliferation, invasion in vitro and growth, metastasis in vivo. In addition, FDX1 affects metabolism of HCC cells and is associated with autophagy. We then confirmed that FDX1 deficiency increases ROS levels, activates mitophagy and the PI3K/AKT signaling pathway in HCC cells. Interestingly, scavenging ROS attenuates the tumor-promoting role and mitophagy of FDX1 downregulation. The results of HTVi and PDO models both find that FDX1 elevation significantly inhibits HCC progression. Moreover, low FDX1 expression is associated with shorter survival and is an independent risk factor for prognosis in HCC patients. CONCLUSIONS: Our research had investigated novel functions of FDX1 in HCC. Downregulation of FDX1 contributes to metabolic reprogramming and leads to ROS-mediated activation of mitophagy and the PI3K/AKT signaling pathway. FDX1 is a potential prognostic biomarker and increasing FDX1 expression may be a potential therapeutic approach to inhibit HCC progression.

The evolutionary arch of bioenergetics from prebiotic mechanisms to the emergence of a cellular respiratory chain.

This article proposes an evolutionary trajectory for the development of biological energy producing systems. Six main stages of energy producing system evolution are described, from early evolutionary pyrite-pulled mechanism through the Last Universal Common Ancestor (LUCA) to contemporary systems. We define the Last Pure Chemical Entity (LPCE) as the last completely non-enzymatic entity. LPCE could have had some life-like properties, but lacked genetic information carriers, thus showed greater instability and environmental dependence than LUCA. A double bubble model is proposed for compartmentalization and cellularization as a prerequisite to both highly efficient protein synthesis and transmembrane ion-gradient. The article finds that although LUCA predominantly functioned anaerobically, it was a non-exclusive anaerobe, and sulfur dominated metabolism preceded phosphate dominated one.

Crystal structure of pectocin M1 reveals diverse conformations and interactions during its initial step via the ferredoxin uptake system.

Pectocin M1 (PM1), the bacteriocin from phytopathogenic Pectobacterium carotovorum which causes soft rot disease, has a unique ferredoxin domain that allows it to use FusA of the plant ferredoxin uptake system. To probe the structure-based mechanism of PM1 uptake, we determined the X-ray structure of full-length PM1, containing an N-terminal ferredoxin and C-terminal catalytic domain connected by helical linker, at 2.04 Å resolution. Based on published FusA structure and NMR data for PM1 ferredoxin domain titrated with FusA, we modeled docking of the ferredoxin domain with FusA. Combining the docking models with the X-ray structures of PM1 and FusA enables us to propose the mechanism by which PM1 undergoes dynamic domain rearrangement to translocate across the target cell outer membrane.

Ferredoxin: A novel antimicrobial peptide derived from the black scraper (Thamnaconus modestus).

Ferredoxin (FDX) is a highly conserved iron-sulfur protein that participates in redox reactions and plays an important role as an electron transport protein in biological processes. However, its function in marine fish remains unclear. We identified two ferrodoxin proteins, FDX1 and FDX2, from black scraper (Thamnaconus modestus) to confirm their genetic structures and expression profiles and to investigate their antimicrobial activity properties by fabricating them with antimicrobial peptides based on sequences. The two TmFDXs mRNAs were most abundant in peripheral blood leukocytes of healthy T. modestus. After artificial infection with Vibrio anguillarum, a major pathogen of T. modestus, TmFDX1 mRNA was significantly upregulated in the gills, heart, intestines, kidneys, liver, and spleen, but was consistently downregulated in the brain. The expression levels of TmFDX2 mRNA were significantly upregulated in the heart, intestines, kidneys, liver, and spleen; however, no significant changes in expression were observed in the brain or gills. Based on the 2Fe-2S ferredoxin-type iron-sulfur-binding domain sequence, two peptides (pFDX1 and pFDX2) were synthesized. The bactericidal effect, biofilm formation inhibition, and gDNA-binding activity of these peptides were investigated. These findings highlight the potential as a natural peptide candidate for TmFDXs.

Iron-sulfur peptides mimicking ferredoxin for an efficient electron transfer to hydrogenase.

In the green alga Chlamydomonas reinhardtii, hydrogenase HydA1 converts protons and electrons to H2 at the H-cluster, which includes a [4Fe-4S] cluster linked to a [2Fe] cluster. The yield of H2 is limited by the electron transfer to HydA1, mediated by the iron-sulfur unit of a photosynthetic electron transfer ferredoxin (PetF). In this study, I have investigated by molecular dynamics and the hybrid quantum mechanics/molecular mechanics method two canonical iron-sulfur peptides (PM1 and FBM) that hold potential as PetF replacements. Using a docking approach, I predict that the distance between the two iron-sulfur clusters in FBM/HydA1 is shorter than in PM1/HydA1, ensuring a greater electron transfer rate. This finding is in line with the reported higher H2 production rates for FBM/HydA1. I also show that the redox potential of these peptides, and therefore their electron transfer properties, can be changed by single-residue mutations in the secondary coordination sphere of their cluster. In particular, I have designed a PM1 variant that disrupts the hydrogen-bonding network between water and the cluster, shifting the redox potential negatively compared to PM1. These results will guide experiments aimed at replacing PetF with peptides that can unlock the biotechnological potential of the alga.

Cuproptosis, the novel type of oxidation-induced cell death in thoracic cancers: can it enhance the success of immunotherapy?

Copper is an important metal micronutrient, required for the balanced growth and normal physiological functions of human organism. Copper-related toxicity and dysbalanced metabolism were associated with the disruption of intracellular respiration and the development of various diseases, including cancer. Notably, copper-induced cell death was defined as cuproptosis which was also observed in malignant cells, representing an attractive anti-cancer instrument. Excess of intracellular copper leads to the aggregation of lipoylation proteins and toxic stress, ultimately resulting in the activation of cell death. Differential expression of cuproptosis-related genes was detected in normal and malignant tissues. Cuproptosis-related genes were also linked to the regulation of oxidative stress, immune cell responses, and composition of tumor microenvironment. Activation of cuproptosis was associated with increased expression of redox-metabolism-regulating genes, such as ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), lipoyltransferase 1 (LIPT1), dihydrolipoamide dehydrogenase (DLD), drolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), and pyruvate dehydrogenase E1 subunit beta (PDHB)). Accordingly, copper-activated network was suggested as an attractive target in cancer therapy. Mechanisms of cuproptosis and regulation of cuproptosis-related genes in different cancers and tumor microenvironment are discussed in this study. The analysis of current findings indicates that therapeutic regulation of copper signaling, and activation of cuproptosis-related targets may provide an effective tool for the improvement of immunotherapy regimens.

Structural aspects of iron­sulfur protein biogenesis: An NMR view.

Over the last decade, structural aspects involving iron­sulfur (Fe/S) protein biogenesis have played an increasingly important role in understanding the high mechanistic complexity of mitochondrial and cytosolic machineries maturing Fe/S proteins. In this respect, solution NMR has had a significant impact because of its ability to monitor transient protein-protein interactions, which are abundant in the networks of pathways leading to Fe/S cluster biosynthesis and transfer, as well as thanks to the developments of paramagnetic NMR in both terms of new methodologies and accurate data interpretation. Here, we review the use of solution NMR in characterizing the structural aspects of human Fe/S proteins and their interactions in the framework of Fe/S protein biogenesis. We will first present a summary of the recent advances that have been achieved by paramagnetic NMR and then we will focus our attention on the role of solution NMR in the field of human Fe/S protein biogenesis.

The crystal structure of methanogen McrD, a methyl-coenzyme M reductase-associated protein.

Methyl-coenzyme M reductase (MCR) is a multi-subunit (α2ß2γ2) enzyme responsible for methane formation via its unique F430 cofactor. The genes responsible for producing MCR (mcrA, mcrB and mcrG) are typically colocated with two other highly conserved genes mcrC and mcrD. We present here the high-resolution crystal structure for McrD from a human gut methanogen Methanomassiliicoccus luminyensis strain B10. The structure reveals that McrD comprises a ferredoxin-like domain assembled into an α + ß barrel-like dimer with conformational flexibility exhibited by a functional loop. The description of the M. luminyensis McrD crystal structure contributes to our understanding of this key conserved methanogen protein typically responsible for promoting MCR activity and the production of methane, a greenhouse gas.

Amino acid residues responsible for the different pH dependency of cell-specific ferredoxins in the electron transfer reaction with ferredoxin-NADP+ reductase from maize leaves.

In the chloroplast stroma, dynamic pH changes occur from acidic to alkaline in response to fluctuating light conditions. We investigated the pH dependency of the electron transfer reaction of ferredoxin-NADP+ reductase (FNR) with ferredoxin (Fd) isoproteins, Fd1 and Fd2, which are localized in mesophyll cells and bundle sheath cells, respectively, in the leaves of C4 plant maize. The pH-dependent profile of the electron transfer activity with FNR was quite different between Fd1 and Fd2, which was mainly explained by the opposite pH dependency of the Km value of these Fds for FNR. Replacement of the amino acid residue at position of 65 (D65N) and 78 (H78A) between the two Fds conferred different effect on their pH dependency of the Km value. Double mutations of the two residues between Fd1 and Fd2 (Fd1D65N/H78A and Fd2N65D/A78H) led to the mutual exchange of the pH dependency of the electron transfer activity. This exchange was mainly explained by the changes in the pH-dependent profile of the Km values. Therefore, the differences in Asp/Asn at position 65 and His/Ala at position 78 between Fd1 and Fd2 were shown to be the major determinants for their different pH dependency in the electron transfer reaction with FNR.

An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Curcuminoid PBPD induces cuproptosis and endoplasmic reticulum stress in cervical cancer via the Notch1/RBP-J/NRF2/FDX1 pathway.

Curcumin has been shown to have antitumor properties, but its low potency and bioavailability has limited its clinical application. We designed a novel curcuminoid, [1-propyl-3,5-bis(2-bromobenzylidene)-4-piperidinone] (PBPD), which has higher antitumor strength and improves bioavailability. Cell counting kit-8 was used to detect cell activity. Transwell assay was used to detect cell invasion and migration ability. Western blot and quantitative polymerase chain reaction were used to detect protein levels and their messenger RNA expression. Immunofluorescence was used to detect the protein location. PBPD significantly inhibited the proliferation of cervical cancer cells, with an IC50 value of 4.16 µM for Hela cells and 3.78 µM for SiHa cells, leading to the induction of cuproptosis. Transcriptome sequencing analysis revealed that PBPD significantly inhibited the Notch1/Recombination Signal Binding Protein for Immunoglobulin kappa J Region (RBP-J) and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathways while upregulating ferredoxin 1 (FDX1) expression. Knockdown of Notch1 or RBP-J significantly inhibited NRF2 expression and upregulated FDX1 expression, leading to the inhibition of nicotinamide adenine dinucleotide phosphate activity and the induction of oxidative stress, which in turn activated endoplasmic reticulum stress and induced cell death. The overexpression of Notch1 or RBP-J resulted in the enrichment of RBP-J within the NRF2 promoter region, thereby stimulating NRF2 transcription. NRF2 knockdown resulted in increase in FDX1 expression, leading to cuproptosis. In addition, PBPD inhibited the acidification of tumor niche and reduced cell metabolism to inhibit cervical cancer cell invasion and migration. In conclusion, PBPD significantly inhibits the proliferation, invasion, and migration of cervical cancer cells and may be a novel potential drug candidate for treatment of cervical cancer.

Redox Properties of Bacillus subtilis Ferredoxin:NADP+ Oxidoreductase: Potentiometric Characteristics and Reactions with Pro-Oxidant Xenobiotics.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

Harnessing iron­sulfur enzymes for synthetic biology.

Reactions catalysed by iron-sulfur (Fe-S) enzymes appear in a variety of biosynthetic pathways that produce valuable natural products. Harnessing these biosynthetic pathways by expression in microbial cell factories grown on an industrial scale would yield enormous economic and environmental benefits. However, Fe-S enzymes often become bottlenecks that limits the productivity of engineered pathways. As a consequence, achieving the production metrics required for industrial application remains a distant goal for Fe-S enzyme-dependent pathways. Here, we identify and review three core challenges in harnessing Fe-S enzyme activity, which all stem from the properties of Fe-S clusters: 1) limited Fe-S cluster supply within the host cell, 2) Fe-S cluster instability, and 3) lack of specialized reducing cofactor proteins often required for Fe-S enzyme activity, such as enzyme-specific flavodoxins and ferredoxins. We highlight successful methods developed for a variety of Fe-S enzymes and electron carriers for overcoming these difficulties. We use heterologous nitrogenase expression as a grand case study demonstrating how each of these challenges can be addressed. We predict that recent breakthroughs in protein structure prediction and design will prove well-suited to addressing each of these challenges. A reliable toolkit for harnessing Fe-S enzymes in engineered metabolic pathways will accelerate the development of industry-ready Fe-S enzyme-dependent biosynthesis pathways.

The extremophilic Andean isolate Acinetobacter sp. Ver3 expresses two ferredoxin-NADP+ reductase isoforms with different catalytic properties.

Ferredoxin/flavodoxin-NADPH reductases (FPRs) catalyze the reversible electron transfer between NADPH and ferredoxin/flavodoxin. The Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes contains two isoenzymes, FPR1ver3 and FPR2ver3. Absorption spectra of these FPRs revealed typical features of flavoproteins, consistent with the use of FAD as a prosthetic group. Spectral differences indicate distinct electronic arrangements for the flavin in each enzyme. Steady-state kinetic measurements show that the enzymes display catalytic efficiencies in the order of 1-6 µm-1·s-1, although FPR1ver3 exhibited higher kcat values compared to FPR2ver3. When flavodoxinver3 was used as a substrate, both reductases exhibited dissimilar behavior. Moreover, only FPR1ver3 is induced by oxidative stimuli, indicating that the polyextremophile Ver3 has evolved diverse strategies to cope with oxidative environments.

EPR studies of ferredoxin in spinach and cyanobacterial thylakoids related to photosystem I-driven NADP+ reduction.

Photosynthetic light-dependent reactions occur in thylakoid membranes where embedded proteins capture light energy and convert it to chemical energy in the form of ATP and NADPH for use in carbon fixation. One of these integral membrane proteins is Photosystem I (PSI). PSI catalyzes light-driven transmembrane electron transfer from plastocyanin (Pc) to oxidized ferredoxin (Fd). Electrons from reduced Fd are used by the enzyme ferredoxin-NADP+ reductase (FNR) for the reduction of NADP+ to NADPH. Fd and Pc are both small soluble proteins whereas the larger FNR enzyme is associated with the membrane. To investigate electron shuttling between these diffusible and embedded proteins, thylakoid photoreduction of NADP+ was studied. As isolated, both spinach and cyanobacterial thylakoids generate NADPH upon illumination without extraneous addition of Fd. These findings indicate that isolated thylakoids either (i) retain a "pool" of Fd which diffuses between PSI and membrane bound FNR or (ii) that a fraction of PSI is associated with Fd, with the membrane environment facilitating PSI-Fd-FNR interactions which enable multiple turnovers of the complex with a single Fd. To explore the functional association of Fd with PSI in thylakoids, electron paramagnetic resonance (EPR) spectroscopic methodologies were developed to distinguish the signals for the reduced Fe-S clusters of PSI and Fd. Temperature-dependent EPR studies show that the EPR signals of the terminal [4Fe-4S] cluster of PSI can be distinguished from the [2Fe-2S] cluster of Fd at > 30 K. At 50 K, the cw X-band EPR spectra of cyanobacterial and spinach thylakoids reduced with dithionite exhibit EPR signals of a [2Fe-2S] cluster with g-values gx = 2.05, gy = 1.96, and gz = 1.89, confirming that Fd is present in thylakoid preparations capable of NADP+ photoreduction. Quantitation of the EPR signals of P700+ and dithionite reduced Fd reveal that Fd is present at a ratio of ~ 1 Fd per PSI monomer in both spinach and cyanobacterial thylakoids. Light-driven electron transfer from PSI to Fd in thylakoids confirms Fd is functionally associated (< 0.4 Fd/PSI) with the acceptor end of PSI in isolated cyanobacterial thylakoids. These EPR experiments provide a benchmark for future spectroscopic characterization of Fd interactions involved in multistep relay of electrons following PSI charge separation in the context of photosynthetic thylakoid microenvironments.