Iron reductase for magnetite synthesis in the magnetotactic bacterium Magnetospirillum magnetotacticum

Ferric iron reductase was purified from magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum (ATCC 31632) to an electrophoretically homogeneous state. The enzyme was loosely bound on the cytoplasmic face of the cytoplasmic membrane and was found more frequently in magnetic cells than in nonmagnetic cells. The molecular mass of the purified enzyme was calculated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 36 kDa, almost the same as that calibrated by gel filtration analysis. The enzyme required NADH and flavin mononucleotide (FMN) as optimal electron donor and cofactor, respectively, and the activity was strongly inhibited by Zn2+ acting as a partial mixed-type inhibitor. The Km values for NADH and FMN were 4.3 and 0. 035 microM, respectively, and the Ki values for Zn2+ were 19.2 and 23.9 microM for NADH and FMN, respectively. When the bacterium was grown in the presence of ZnSO4, the magnetosome number in the cells and the ferric iron reductase activity declined in parallel with an increase in the ZnSO4 concentration of the medium, suggesting that the ferric iron reductase purified in the present study may participate in magnetite synthesis.     

Iron reductase activity in cowpea Rhizobium GN1 (peanut isolate)

Cell-free extracts of cowpea Rhizobium GN1 (peanut isolate) possessed reductase activity towards various ferric siderophores including its own. This activity was heat- and O2-sensitive and required NADH and flavins. Whereas NADPH could replace NADH with half-efficiency, succinate could not serve as reductant. Activity was insensitive to antimycin A and rotenone but was completely inhibited by HgCl2 (1 mM). Mg2+ (1 mM) enhanced reductase activity but Zn2+ and Cu2+ at the same concentration were inhibitory. The enzyme was located in the periplasmic fraction.     

Role of molybdate and other transition metals in the accumulation of protochelin by Azotobacter vinelandii

Both molybdate and iron are metals that are required by the obligately aerobic organism Azotobacter vinelandii to survive in the nutrient-limited conditions of its natural soil environment. Previous studies have shown that a high concentration of molybdate (1 mM) affects the formation of A. vinelandii siderophores such that the tricatecholate protochelin is formed to the exclusion of the other catecholate siderophores, azotochelin and aminochelin. It has been shown previously that molybdate combines readily with catecholates and interferes with siderophore function. In this study, we found that the manner in which each catecholate siderophore interacted with molybdate was consistent with the structure and binding potential of the siderophore. The affinity that each siderophore had for molybdate was high enough that stable molybdo-siderophore complexes were formed but low enough that the complexes were readily destabilized by Fe(3+). Thus, competition between Fe(3+) and molybdate did not appear to be the primary cause of protochelin accumulation; in addition, we determined that protochelin accumulated in the presence of vanadate, tungstate, Zn(2+), and Mn(2+). We found that all five of these metal ions partially inhibited uptake of (55)Fe-protochelin and (55)Fe-azotochelin complexes. Also, each of these metal ions partially inhibited the activity of ferric reductase, an enzyme important in the deferration of ferric siderophores. Our results suggest that protochelin accumulates in the presence of molybdate because protochelin uptake and conversion into its component parts, azotochelin and aminochelin, are inhibited by interference with ferric reductase.     

Reduction of ferric citrate catalyzed by NADH:nitrate reductase

We show that NADH:nitrate reductase from squash cotyledons can catalyze the reduction of ferric citrate. When nitrate reductase was purified to homogeneity using a two-step affinity chromatography procedure, an NADH:Fe(III)-citrate reductase activity copurified with it and had identical electrophoretic mobility to it. The iron reductase activity was optimum near pH 6.3, had an apparent Km for Fe(III)-citrate of 0.02 mM, and was inhibited by monospecific anti-nitrate reductase rabbit sera. Differential inhibition of the enzyme's activities indicated iron and nitrate were reduced at different sites. In addition to its role in nitrogen assimilation, nitrate reductase catalyzes ferric citrate reduction and could have a role in iron assimilation.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

Physiological and biochemical alterations in Anabaena 7120 under iron stress

Various physiological and biochemical process like growth, NO3- -uptake, nitrate reductase, glutamine synthetase and ATPases (Mg2+ and Ca2+ dependent) in the cyanobacterium Anabaena 7120 were observed under iron stress. Growth was found to be maximum in 50 microM Fe3+ added cells however, 20 microM Fe3+ (the Fe3+ concentration generally used for routine culturing of cyanobacterial cell in Chu 10 medium) incubation resulted in lower growth. Fe3+ starvation on the other hand showed very poor growth up to 4th day but once the growth started it reached at significant level on 7th day. Higher Fe3+ concentration reflected reduced growth with lethality at 500 microM Fe3+. Chlorophyll a fluorescence under Fe3+ stress reflected almost the similar results as in case of growth. However, the pigment was found to be more sensitive as compared to protein under Fe3+ stress. Similar results have been observed in case of NO3-uptake with only 80% reduction in nutrient uptake in 500 microM Fe3+ incubated cells. Nitrate reductase activity was lower in Fe3+ starved cells as compared to significant enzyme activity in 20 and 50 microM Fe3+ incubated cells. Similar to nitrate reductase, glutamine synthetase also showed maximum level in 50 microM Fe3+ added cells, however, higher Fe3+ concentration (300-500 microM ) resulted in reduced enzymatic activity. Glutamine synthetase activity was less sensitivity as compared to nitrate reductase activity under Fe3+ stress. ATPase (Mg2+ and Ca2+ dependent) always showed higher level with increasing Fe3+ concentration.     

Chromate reduction by cell-free extract of Bacillus firmus KUCr1

Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.     

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.     

Identification and characterization of a novel ferric reductase from the hyperthermophilic Archaeon Archaeoglobus fulgidus

Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe(3+)-EDTA reductase activity in its soluble protein fraction. The corresponding enzyme, which constitutes about 0.75% of the soluble protein, was purified 175-fold to homogeneity. Based on SDS-polyacrylamide gel electrophoresis, the ferric reductase consists of a single subunit with a M(r) of 18,000. The M(r) of the native enzyme was determined by size exclusion chromatography to be 40,000 suggesting that the native ferric reductase is a homodimer. The enzyme uses both NADH and NADPH as electron donors to reduce Fe(3+)-EDTA. Other Fe(3+) complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe(3+) is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe(3+) reduction. Ferric reductase also reduces FMN and FAD, but not riboflavin, with NAD(P)H which classifies the enzyme as a NAD(P)H:flavin oxidoreductase. The enzyme exhibits a temperature optimum of 88 degrees C. When incubated at 85 degrees C, the enzyme activity half-life was 2 h. N-terminal sequence analysis of the purified ferric reductase resulted in the identification of the hypothetical gene, AF0830, of the A. fulgidus genomic sequence. The A. fulgidus ferric reductase shares amino acid sequence similarity with a family of NAD(P)H:FMN oxidoreductases but not with any ferric reductases suggesting that the A. fulgidus ferric reductase is a novel enzyme.     

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.     

Cell-free mercury volatilization activity from three marine caulobacter strains

Three mercury-resistant marine Caulobacter strains showed an inducible mercury volatilization activity. Cell-free mercury volatilization (mercuric reductase) from these three marine Caulobacter strains was characterized and compared with enzyme activities determined by plasmids of Escherichia coli and Staphylococcus aureus. The temperature sensitivity of the Caulobacter mercuric reductase was greater than that of mercuric reductase from other gram-negative sources. Cell-free enzyme activity required NADH or NADPH, with NADPH functioning much better at lower concentrations than NADH. The Km for the Caulobacter enzyme was 4 microM Hg2+. Ag+ was a competitive inhibitor of Caulobacter mercuric reductase (Ki = 0.2 microM Ag+), as with previously studied enzymes. Arsenite was a noncompetitive inhibitor of the Caulobacter enzyme with a Ki of 75 microM AsO2-.     

Cell-free mercury volatilization activity from three marine caulobacter strains.

Three mercury-resistant marine Caulobacter strains showed an inducible mercury volatilization activity. Cell-free mercury volatilization (mercuric reductase) from these three marine Caulobacter strains was characterized and compared with enzyme activities determined by plasmids of Escherichia coli and Staphylococcus aureus. The temperature sensitivity of the Caulobacter mercuric reductase was greater than that of mercuric reductase from other gram-negative sources. Cell-free enzyme activity required NADH or NADPH, with NADPH functioning much better at lower concentrations than NADH. The Km for the Caulobacter enzyme was 4 microM Hg2+. Ag+ was a competitive inhibitor of Caulobacter mercuric reductase (Ki = 0.2 microM Ag+), as with previously studied enzymes. Arsenite was a noncompetitive inhibitor of the Caulobacter enzyme with a Ki of 75 microM AsO2-.     

Plasma membrane-bound NADH: Fe3+-EDTA reductase and iron deficiency in tomato (Lycopersicon esculentum). Is there a Turbo reductase?

The properties of NADH-dependent Fe³⁺-EDTA reductase in plasma membranes (PM) from roots of iron-deficient and -sufficient tomato plants [Lycopersicon esculentum L. (Mill.) cv. Abunda] were examined. Iron deficiency resulted in a 3-fold increase of in vivo root iron-chelate reductase activity with a K_m (Fe³⁺-EDTA) of 230 μM. In purified root PM, average specific activities of ferric chelate reductase of 410 and 254 nmol Fe (mg protein)^?1 min^?1 were obtained for iron-deficient and -sufficient plants, respectively. In both cases, the PM-bound activity showed a pH optimum at pH 6.8. Activity depended on NADH and not on NADPH and on the presence of detergent. The activity was inhibited 40-50% by superoxide dismutase (EC 1.15.1.1) and ca 30% by oxygen. Kinetic analysis of the membrane-bound enzyme revealed a K_m (Fe³⁺-EDTA) of ca 200 μM for both iron-stressed and -sufficient plants. For NADH, K_m values around 230 μM were obtained. The ferric chelate reductase could be solubilised from salt-washed PM with Triton X-100 at a protein:detergent ratio of 1:2.8 (w/w). The Triton-soluble fraction revealed one enzyme-stained band in native polyacrylamide electrophoresis. Although the membranes showed no nitrate reductase (NR; EC 1.6.6.1) activity, anti-spinach NR immunoglobulin G (IgG) recognized a 54 kDa band both in the PM and the Triton-soluble fraction, but not in the enzymatically active material obtained from the native gel. No evidence could be found for the synthesis of a new, biochemically distinct PM-bound ferric chelate reductase under iron deficiency, which might be identified as the so-called Turbo reductase. It is concluded that iron deficiency in tomato induces increased expression of a ferric chelate reductase in root PM, which is already present in iron-sufficient plants and probably also in plants, which do not contain the Turbo reductase, like the grasses. The iron reductase is not identical with the recently reported PM-associated nitrate reductase.     

Ferric leghemoglobin reductase from soybean root nodules

An NADH: (acceptor) oxidoreductase from the cytosol of soybean root nodules was purified by ammonium sulfate fractionation, hydroxylapatite adsorption, and Sephacryl S-200 Superfine chromatography. The native molecular weight of the reductase was found to be 100,000 by analytical gel filtration and 83,000 by equilibrium ultracentrifugation. The subunit molecular weight was 54,000 as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. With ferric leghemoglobin (Lb) as the substrate, nearly identical initial velocities were obtained using either CO or O2 to ligate the enzymatically produced ferrous leghemoglobin. With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of ferric Lb was spectrally identified as LbCO. Initial velocity was a linear function of increasing enzyme concentration. NADPH was only 31% as effective an electron donor as NADH as determined by initial velocity. The Michaelis constants (Km) for ferric Lba and NADH were 9.5 and 18.8 microM, respectively. Myoglobin, Lba, Lbc1, Lbc2, Lbc3, and Lbd were reduced at similar rates by the reductase. At pH 5.2, acetate-bound ferric Lb and nicotinate-bound ferric Lb were reduced by the enzyme at 83 and 5%, respectively, of rates observed in the absence of these ligands. The rate of enzymatic reduction of ferric Lb was constant between pH 6.5 and 7.6 but increased approximately threefold at pH 5.2. The results indicate that the NADH: (acceptor) oxidoreductase could be identified as a ferric Lb reductase.     

The identification and biosynthesis of siderochromes formed by Micrococcus denitrificans.

1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.     

Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD

A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 degrees C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group.     

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP⁺ reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k _cat/K _m value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k _cat/K _m value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.     

Ferric and cupric reductase activities by iron-limited cells of the green alga Chlorella kessleri: quantification via oxygen electrode

The colorimetric Fe2+ indicators bathophenanthroline disulfonic acid (BPDS) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FZ) are routinely used to assay for plasma membrane ferric reductase activity in iron-limited algal cells and also in roots from iron-limited plants. Ferric reductase assays using these colorimetric indicators must take into account the fact that Fe3+ chelators (e.g. ethylenediaminetetraacetic acid) can also in general bind Fe2+ and may therefore compete with the colorimetric Fe2+ indicators, leading to the potential for underestimation of the ferric reduction rate. Conversely, the presence of BPDS or FZ may also facilitate the reduction of Fe3+ chelates, potentially leading to overestimation of ferric reduction rates. Last, both BPDS and FZ have non-negligible affinities for Fe3+ in addition to their well-known affinities for Fe2+; this leads to potential difficulties in ascertaining whether free and/or chelated Fe3+ are potential substrates for the ferric reductase. Similar issues arise when assaying for cupric reductase activity using the colorimetric Cu+ indicator bathocuproinedisulfonic acid (BCDS). In this paper, we describe an oxygen-electrode-based assay (conducted in darkness) for both ferric and cupric reductase activities that does not use colorimetric indicators. Using this assay system, we show that the plasma membrane metal reductase activity of iron-limited cells of the green alga Chlorella kessleri reduced complexed Fe3+ (i.e. Fe3+ chelates) but did not reduce free (non-chelated) Fe3+, and also reduced free Cu2+ to Cu+, but did not reduce Cu2+ that was part of Cu2+ chelates. We suggest that the potential for reduction of free Fe3+ cannot be adequately assayed using colorimetric assays. As well, the BPDS-based assay system consistently yielded similar estimates of ferric reductase activity compared with the O2-electrode-based assays at relatively low Fe3+ concentration, but higher estimates at higher Fe3+ concentrations with chelators other than desferrioxamine mesylate. With respect to cupric reductase activity, the O2 electrode consistently provided much higher estimates; we suggest that this was as a result of Cu2+ chelation by BCDS leading to a large underestimation of the true cupric reduction rate. These results suggest that an O2-electrode-based metal reductase assay system has some specific advantages compared with the traditional colorimetric assay system, including especially the ability to discriminate between the reduction of free metal ions and chelated metal ions.     

Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase.

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.