Copper induces neuron-sparing, ferredoxin 1-independent astrocyte toxicity mediated by oxidative stress.

Copper is an essential enzyme cofactor in oxidative metabolism, anti-oxidant defenses, and neurotransmitter synthesis. However, intracellular copper, when improperly buffered, can also lead to cell death. Given the growing interest in the use of copper in the presence of the ionophore elesclomol (CuES) for the treatment of gliomas, we investigated the effect of this compound on the surround parenchyma-namely neurons and astrocytes in vitro. Here, we show that astrocytes were highly sensitive to CuES toxicity while neurons were surprisingly resistant, a vulnerability profile that is opposite of what has been described for zinc and other toxins. Bolstering these findings, a human astrocytic cell line was similarly sensitive to CuES. Modifications of cellular metabolic pathways implicated in cuproptosis, a form of copper-regulated cell death, such as inhibition of mitochondrial respiration or knock-down of ferredoxin 1 (FDX1), did not block CuES toxicity to astrocytes. CuES toxicity was also unaffected by inhibitors of apoptosis, necrosis or ferroptosis. However, we did detect the presence of lipid peroxidation products in CuES-treated astrocytes, indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Indeed, treatment with anti-oxidants mitigated CuES-induced cell death in astrocytes indicating that oxidative stress is a mediator of CuES-induced glial toxicity. Lastly, prior induction of metallothioneins 1 and 2 in astrocytes with zinc plus pyrithione was strikingly protective against CuES toxicity. As neurons express high levels of metallothioneins basally, these results may partially account for their resistance to CuES toxicity. These results demonstrate a unique toxic response to copper in glial cells which contrasts with the cell selectivity profile of zinc, another biologically relevant metal.

Dexmedetomidine enables copper homeostasis in cerebral ischemia/reperfusion via ferredoxin 1.

Excessive oxygen free radicals and toxic substances are generated in cerebral ischemia-reperfusion (I/R) process. Dexmedetomidine (DEX), a common anesthetic and sedative drug, can considerably boost glutathione (GSH), which has anti-copper influx effects. Focusing on cuproptosis, the mechanism of DEX in the I/R was revealed. Using the I/R rat model, the effects of DEX and the copper chelator D-penicillamine on cerebral infarct volume, copper levels, mitochondrial respiration and membrane potential, GSH content, and enrichment of cuproptosis functional proteins were examined. The involvement of ferredoxin 1 (FDX1) in the DEX regulatory pathway was verified by overexpressing FDX1 in vitro. DEX could significantly reduce cerebral infarction in rats, reduce copper levels, maintain mitochondrial functions, increase GSH, and reduce the content of key proteins related to cuproptosis. These aspects were replicated in vitro and revealed that FDX1 overexpression partially reversed the impacts of DEX. Together, cuproptosis occurs in the brain I/R process and DEX can enhance cell survival by blocking the primary pathway mediated by FDX1.KEY MESSAGESDexmedetomidine reduces cerebral infarction in the I/R rat models.Dexmedetomidine reduces cuproptosis in the I/R rat models.FDX1, an upstream of protein fatty acylation, mediates regulation of Dexmedetomidine.

Ferredoxin reductase regulates proliferation, differentiation, cell cycle and lipogenesis but not apoptosis in SZ95 sebocytes.

Ferredoxin reductase (FDXR), a mitochondrial membrane-associated flavoprotein, is essential for electron transfer and modulates p53-dependent apoptosis in cancer cells.FDXR may be implicated in epidermal and sebocytic differentiation, but its explicit function in sebocytes remains to be elucidated. In the present study, immunohistochemistry revealed that FDXR expression was increased in sebaceous cells of acne lesions. FDXR, PPARγ, LXRα/ß, SREBP1 and Sox9 expression was incremental during sebocyte differentiation. FDXR overexpression induced by Ad-GFP-FDXR infection enhanced differentiation, reactive oxygen species (ROS), lipogenesis and PPARγ expression, and consequnently inhibited proliferation in SZ95 sebocytes. Flow cytometry showed that FDXR overexpression induced significant blockade of G2/M phase but had no effect on sub-G1 (apoptotic) sebocytes. Insulin-like growth factor-1 (IGF-1)-induced FDXR and PPARγ expression and lipogenesis were abolished by pretreatment with PI3K inhibitor LY294002. These results suggest that FDXR overexpression might promote differentiation and lipogenesis via ROS production and suppress proliferation via G2/S blockade in SZ95 sebocytes. IGF-1 could facilitate differentiation and lipogenesis through PI3K/Akt/FDXR pathway. FDXR could serve as a potential marker of advanced sebaceous differentiation, and its overexpression may be involved in the development of acne lesions.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin.

Cytochrome P450s are diverse and powerful catalysts that can activate molecular oxygen to oxidize a wide variety of substrates. Catalysis relies on effective uptake of two electrons and two protons. For cytochrome P450cam, an archetypal member of the superfamily, the second electron must be supplied by the redox partner putidaredoxin (Pdx). Pdx also plays an effector role beyond electron transfer, but after decades the mechanism remains under investigation. We applied infrared spectroscopy to heme-ligated CN- to examine the influence of Pdx binding. The results indicate that Pdx induces the population of a conformation wherein the CN- ligand forms a strong hydrogen bond to a solvent water molecule, experimentally corroborating the formation of a proposed proton delivery network. Further, characterization of T252A P450cam implicates the side chain of Thr252 in regulating the population equilibrium of hydrogen-bonded states within the P450cam/Pdx complex, which could underlie its role in directing activated oxygen toward product formation and preventing reaction uncoupling through peroxide release.

Ferredoxin-mediated reduction of 2-nitrothiophene inhibits photosynthesis: mechanism and herbicidal potential.

Searching for compounds that inhibit the growth of photosynthetic organisms highlighted a prominent effect at micromolar concentrations of the nitroheteroaromatic thioether, 2-nitrothiophene, applied in the light. Since similar effects were reminiscent to those obtained also by radicals produced under excessive illumination or by herbicides, and in light of its redox potential, we suspected that 2-nitrothiophene was reduced by ferredoxin, a major reducing compound in the light. In silico examination using docking and tunneling computing algorithms of the putative interaction between 2-nitrothiophene and cyanobacterial ferredoxin has suggested a site of interaction enabling robust electron transfer from the iron-sulfur cluster of ferredoxin to the nitro group of 2-nitrothiophene. ESR and oximetry analyses of cyanobacterial cells (Anabaena PCC7120) treated with 50 µM 2-nitrothiophene under illumination revealed accumulation of oxygen radicals and peroxides. Gas chromatography mass spectrometry analysis of 2-nitrothiophene-treated cells identified cytotoxic nitroso and non-toxic amino derivatives. These products of the degradation pathway of 2-nitrohiophene, which initializes with a single electron transfer that forms a short-live anion radical, are then decomposed to nitrate and thiophene, and may be further reduced to a nitroso hydroxylamine and amino derivatives. This mechanism of toxicity is similar to that of nitroimidazoles (e.g. ornidazole and metronidazole) reduced by ferredoxin in anaerobic bacteria and protozoa, but differs from that of ornidazole in planta.

Antimicrobial properties of acrylic resins doped with Undaria pinnatifida exposed to light-emitting diode: In silico and in vitro assessments on multispecies biofilm-producing microbiota.

BACKGROUND: This study sought to evaluates the efficiency of anti-microbial activity of acrylic resins doped with different concentrations of Undaria pinnatifida after activation with light-emitting diode (LED) at producing photodynamic damage to multispecies biofilm-producing microbiome. MATERIAL AND METHODS: In this study, bioinformatics tools and computer simulation molecular modeling were used to evaluate the capacity of ferredoxin (FDX), an electron acceptor in metabolic pathways of U. pinnatifida, which can discharge electrons produced from photo-excited chlorophyll-a (Chl-a) by LED irradiation. Acrylic resin discs containing different concentration of U. pinnatifida (0, 0.5, 1, and 2%) were fabricated and were subjected to LED irradiation immediately before each experiment. After continuously rinsed (up to 30 days), the antimicrobial activity of acrylic resins doped with U. pinnatifida following photo-activation was determined by disc agar diffusion, biofilm formation inhibition, and eluted component assays versus bacterial species linked to caries that constitute a mixed biofilm including Streptococcus mutans, S. sanguinis, and Lactobacillus acidophilus, as well as Candida albicans as main etiology of candidal stomatitis. RESULTS: Modeling and a virtual screening analysis of FDX indicated that it is a stable protein with an iron-sulfur center that can discharge electrons produced from photo-excited Chl-a and transfers them to FDX-NADP+ reductase for NADP+ reduction in photosystem I, which is essential in the Calvin cycle for carbon assimilation. FDX acts as an electron transfer agent in the redox reactions. The results showed that growth inhibition zones were not seen around acrylic resin discs in any group. In biofilm test, the colony counts of all test microorganisms significantly decreased (36%-87%) by an increase in the percentage of U. pinnatifida in acrylic resins after photo-activation (P < 0.05). Acrylic resins doped with 2% wt. U. pinnatifida following photo-activation using LED was inhibited biofilm formation by the test microorganisms, up to 30 days of rinsing. CONCLUSION: Based on the results presented here, an acrylic resin containing U. pinnatifida, even at the lowest concentration, following photo-activation using LED have antimicrobial properties against planktonic and biofilm forms of the cariogenic microorganisms as well as C. albicans.

Non-photochemical reduction of thylakoid photosynthetic redox carriers in vitro: relevance to cyclic electron flow around photosystem I?

Non-photochemical (dark) increases in chlorophyll a fluorescence yield associated with non-photochemical reduction of redox carriers (Fnpr) have been attributed to the reduction of plastoquinone (PQ) related to cyclic electron flow (CEF) around photosystem I. In vivo, this rise in fluorescence is associated with activity of the chloroplast plastoquinone reductase (plastid NAD(P)H: plastoquinone oxidoreductase) complex. In contrast, this signal measured in isolated thylakoids has been attributed to the activity of the protein gradient regulation-5 (PGR5)/PGR5-like (PGRL1)-associated CEF pathway. Here, we report a systematic experimentation on the origin of Fnpr in isolated thylakoids. Addition of NADPH and ferredoxin to isolated spinach thylakoids resulted in the reduction of the PQ pool, but neither its kinetics nor its inhibitor sensitivities matched those of Fnpr. Notably, Fnpr was more rapid than PQ reduction, and completely insensitive to inhibitors of the PSII QB site and oxygen evolving complex as well as inhibitors of the cytochrome b6f complex. We thus conclude that Fnpr in isolated thylakoids is not a result of redox equilibrium with bulk PQ. Redox titrations and fluorescence emission spectra imply that Fnpr is dependent on the reduction of a low potential redox component (Em about − 340 mV) within photosystem II (PSII), and is likely related to earlier observations of low potential variants of QA within a subpopulation of PSII that is directly reducible by ferredoxin. The implications of these results for our understanding of CEF and other photosynthetic processes are discussed.

C-terminal region of plant ferredoxin-like protein is required to enhance resistance to bacterial disease in Arabidopsis thaliana.

Protein phosphorylation is an important biological process associated with elicitor-induced defense responses in plants. In a previous report, we described how plant ferredoxin-like protein (PFLP) in transgenic plants enhances resistance to bacterial pathogens associated with the hypersensitive response (HR). PFLP possesses a putative casein kinase II phosphorylation (CK2P) site at the C-terminal in which phosphorylation occurs rapidly during defense response. However, the contribution of this site to the enhancement of disease resistance and the intensity of HR has not been clearly demonstrated. In this study, we generated two versions of truncated PFLP, PEC (extant CK2P site) and PDC (deleted CK2P site), and assessed their ability to trigger HR through harpin (HrpZ) derived from Pseudomonas syringae as well as their resistance to Ralstonia solanacearum. In an infiltration assay of HrpZ, PEC intensified harpin-mediated HR; however, PDC negated this effect. Transgenic plants expressing these versions indicate that nonphosphorylated PFLP loses its ability to induce HR or enhance disease resistance against R. solanacearum. Interestingly, the CK2P site of PFLP is required to induce the expression of the NADPH oxidase gene, AtrbohD, which is a reactive oxygen species producing enzyme. This was further confirmed by evaluating the HR on NADPH oxidase in mutants of Arabidopsis. As a result, we have concluded that the CK2P site is required for the phosphorylation of PFLP to enhance disease resistance.

Reaction pathways involved in the production of hydroxyl radicals in thylakoid membrane: EPR spin-trapping study.

It has been suggested that both free metals and reduced ferredoxin (Fd) participate in the light-induced production of hydroxyl radicals (OH*) in thylakoid membranes of chloroplasts. The most direct evidence for the involvement of Fd in OH* formation under physiological conditions was reported by Jakob and Heber (Plant Cell Physiol., 1996, 37, 629-635), who used the oxidation of dimethylsulfoxide to methane sulfinic acid as an indicator of OH* production. We confirmed their conclusions using a more sensitive and reliable EPR spin-trapping method and extended their work by additional findings. Free metal-dependent and ferredoxin-dependent OH* production was studied simultaneously and strong metal chelator Desferal was used to distinguish between these reaction pathways. The participation of protein-bound iron within photosystem I was confirmed by partial suppression of OH* generation in broken chloroplasts by methyl viologen. The enhancement in the production of OH* in thylakoid membranes by externally added ferredoxin can be considered as a straightforward evidence of the involvement of ferredoxin in OH* formation.

Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar.

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6% complexes of 2-mercapthoethanol (2ME), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpin(pss)-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS).

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin(pss), an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin(pss)-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coli was able to significantly increase active oxygen species (AOS) generation when mixed with harpin(pss) in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin(pss) in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflp gene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin(pss)-mediated hypersensitive cell death in vivo. In response to harpin(pss), suspension cells derived from Pflp transgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl3) were used to study the involvement of AOS in harpin(pss)-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin(pss) inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.

Stearoyl-acyl carrier protein and unusual acyl-acyl carrier protein desaturase activities are differentially influenced by ferredoxin.

Acyl-acyl carrier protein (ACP) desaturases function to position a single double bond into an acyl-ACP substrate and are best represented by the ubiquitous Delta9 18:0-ACP desaturase. Several variant acyl-ACP desaturases have also been identified from species that produce unusual monoenoic fatty acids. All known acyl-ACP desaturase enzymes use ferredoxin as the electron-donating cofactor, and in almost all previous studies the photosynthetic form of ferredoxin rather than the non-photosynthetic form has been used to assess activity. We have examined the influence of different forms of ferredoxin on acyl-ACP desaturases. Using combinations of in vitro acyl-ACP desaturase assays and [(14)C]malonyl-coenzyme A labeling studies, we have determined that heterotrophic ferredoxin isoforms support up to 20-fold higher unusual acyl-ACP desaturase activity in coriander (Coriandrum sativum), Thunbergia alata, and garden geranium (Pelargonium x hortorum) when compared with photosynthetic ferredoxin isoforms. Heterotrophic ferredoxin also increases activity of the ubiquitous Delta9 18:0-ACP desaturase 1.5- to 3.0-fold in both seed and leaf extracts. These results suggest that ferredoxin isoforms may specifically interact with acyl-ACP desaturases to achieve optimal enzyme activity and that heterotrophic isoforms of ferredoxin may be the in vivo electron donor for this reaction.

Interaction of positively charged amino acid residues of recombinant, cyanobacterial ferredoxin:NADP+ reductase with ferredoxin probed by site directed mutagenesis.

The petH genes encoding ferredoxin:NADP+ reductase (FNR) from two Anabaena species (PCC 7119 and ATCC 29413) were cloned and overexpressed in E. coli. Several positively charged residues (Arg, Lys) have been implicated to be involved in ferredoxin binding and electron transfer by cross-linking, chemical modification and protection experiments, and crystallographic studies. The following substitutions were introduced by site-directed mutagenesis: R153Q, K209Q, K212Q, R214Q, K275N, K430Q and K431Q in Anabaena 29413 FNR, and R153E, K209E, K212E, R214E, K275E, R401E, K427E, and K431E in Anabaena 7119 FNR. Comparison of the diaphorase activities, the specific rates of ferredoxin dependent NADP(+)-photoreduction and cytochrome c reduction catalyzed by FNR showed that all these amino acid residues were required for efficient electron transfer between FNR and ferredoxin. Replacement of any one of these basic residues produced a much more pronounced effect on the cytochrome c reductase activity, where FNR, reduced by NADPH, acted as electron donor, than in the reduction of NADP+ by photosystem I via FNR. A mutation involving the replacement of positive charge by a neutral amide produced in all cases a smaller inhibitory effect on the activity than a charge reversal mutation. In addition, it has been found that R214 was necessary for stable integration of the non covalently bound FAD-cofactor.

Phycobilin biosynthesis: reductant requirements and product identification for heme oxygenase from Cyanidium caldarium.

Algal heme oxygenase is a soluble enzyme from Cyanidium caldarium that catalyzes the first committed step of phycobilin biosynthesis by converting protoheme to biliverdin IX alpha. Although the physiological substrate (protoheme) of algal heme oxygenase is identical to that of microsomal heme oxygenase, which catalyzes heme catabolism in animals, the two enzyme systems differ in several respects including the nature of the required reductants and solubility of the enzymes. Addition of the strong Fe3+ ion chelators, desferrioxamine and Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), greatly increased the yield of solvent-extracted bilin product. The effect of the Fe3+ chelators was approximately equal whether they were added during or after the enzyme incubation. Postincubation treatment of the enzyme reaction mixture with strong acid also greatly increased the product yield. Addition of desferrioxamine to the reaction mixture after the incubation was terminated caused the appearance of an absorption spectrum, indicating an increase in the concentration of free bilin product. Acid and Fe3+ chelators are known to cause dissociation of Fe(III)-bilin complexes. These results indicate that the in vitro enzymic reaction product of algal heme oxygenase is a nonenzyme-bound Fe(III)-biliverdin IX alpha complex that is poorly extracted and/or quantitated unless it is first dissociated. Algal heme oxygenase required the simultaneous presence of both reduced ferredoxin and a second reductant such as ascorbate for activity. The requirement for L-ascorbate could be substituted by Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or D-ascorbate, but not by dehydroascorbate or dithiothreitol. Heme oxygenase was purified over 200-fold from C. caldarium by differential (NH4)2SO4 precipitation and serial column chromatography over reactive blue 2-Sepharose, DEAE-cellulose, Sephadex G-75, and ferredoxin-Sepharose.

Ferredoxin-dependent CO2 fixation in bean sprouts.

Extracts of bean sprouts are capable of reducing ferredoxin and of catalyzing the incorporation of bicarbonate and acetyl coenzyme A into an organic compound that is likely to be pyruvate, in a reaction that requires reduced ferredoxin. The rate of the reaction, the first known for which ferredoxin appears to serve as the direct reductant for CO2 fixation in a higher plant, depends on the concentrations of both ferredoxin and bicarbonate, with half-maximal rates being observed at ferredoxin and bicarbonate concentrations of 0.8 microM and 200 microM, respectively.

Cloning and sequencing of the gene encoding spinach ferredoxin-dependent glutamate synthase.

The nucleotide sequences of two cDNA clones, totalling 4948 bp in length, encoding 98% of the 1483 amino acids of the mature form of the ferredoxin-dependent glutamate synthase of spinach chloroplasts have been determined. The amino-terminal sequence of the enzyme has been determined by direct sequencing of the protein. The deduced amino-acid sequence of the spinach enzyme is 83% identical to that of the ferredoxin-dependent maize enzyme and shows significant sequence homology to two prokaryotic NAD(P)H-dependent glutamate synthases. Analysis of spinach genomic DNA indicates the presence of a single-copy gene for the spinach enzyme. Northern analysis reveals the presence of a single 5.5 kb transcript, which is present in higher levels in young spinach leaves than in older leaves.

Comparison of the binding sites of plant ferredoxin for two ferredoxin-dependent enzymes.

Differential chemical modification of acidic residues was used to map the binding site of plant ferredoxin (Fd) for the chloroplast enzyme ferredoxin:thioredoxin reductase (FTR). Binding of FTR to Fd inhibits chemical modification of Fd residues D34, D65, E92, E93, E94 and C-terminal A97. The binding site demarcated by these residues differs from that for ferredoxin:NADP+ reductase (FNR). The FTR site includes C-terminal residues but not helix 24-31, which is part of the FNR site. Both sites enclose the [2Fe-2S] cluster.

In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.

The meta-cleavage operon of the TOL plasmid pWW0 of Pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to Krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. The functions of all the genes are known with the exception of xylT. We constructed pWW0 mutants defective in the xylT gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. In the xylT mutants, all the meta-cleavage enzymes were induced by p-toluate with the exception of catechol 2,3-dioxygenase whose activity was 1% of the p-toluate-induced activity in wild-type cells. Addition of 4-methylcatechol to m-toluate-grown wild-type and xylT cells resulted in the inactivation of catechol 2,3-dioxygenase in these cells. In the wild-type strain but not in the xylT mutant, the catechol 2,3-dioxygenase activity was regenerated in a short time. The regeneration of the catechol 2,3-dioxygenase activity was also observed in H2O2-treated wild-type cells, but not in H2O2-treated xylT cells. We concluded that the xylT product is required for the regeneration of catechol 2,3-dioxygenase.

Purification and properties of methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila.

Methyl coenzyme M methylreductase from acetate-grown Methanosarcina thermophila TM-1 was purified 16-fold from a cell extract to apparent homogeneity as determined by native polyacrylamide gel electrophoresis. Ninety-four percent of the methylreductase activity was recovered in the soluble fraction of cell extracts. The estimated native molecular weight of the enzyme was between 132,000 (standard deviation [SD], 1,200) and 141,000 (SD, 1,200). Denaturing polyacrylamide gel electrophoresis revealed three protein bands corresponding to molecular weights of 69,000 (SD, 1,200), 42,000 (SD, 1,200), and 33,000 (SD, 1,200) and indicated a subunit configuration of alpha 1 beta 1 gamma 1. As isolated, the enzyme was inactive but could be reductively reactivated with titanium (III) citrate or reduced ferredoxin. ATP stimulated enzyme reactivation and was postulated to be involved in a conformational change of the inactive enzyme from an unready state to a ready state that could be reductively reactivated. The temperature and pH optima for enzyme activity were 60 degrees C and between 6.5 and 7.0, respectively. The active enzyme contained 1 mol of coenzyme F430 per mol of enzyme (Mr, 144,000). The Kms for 2-(methylthio)ethane-sulfonate and 7-mercaptoheptanoylthreonine phosphate were 3.3 mM and 59 microM, respectively.