Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity

The arsH gene or its homologs are a frequent part of the arsenic resistance system in bacteria and eukaryotes. Although a specific biological function of the gene product is unknown, the ArsH protein was annotated as a member of the NADPH-dependent FMN reductase family based on a conserved (T/S)XRXXSX(T/S) fingerprint motif common for FMN binding proteins. Presented here are the first crystal structure of an ArsH protein from Shigella flexneri refined at 1.7 A resolution and results of enzymatic activity assays that revealed a strong NADPH-dependent FMN reductase and low azoreductase activities. The ArsH apo protein has an alpha/beta/alpha-fold typical for FMN binding proteins. The asymmetric unit consists of four monomers, which form a tetramer. Buried surface analysis suggests that this tetramer is likely to be the relevant biological assembly. Dynamic light scattering experiments are consistent with this hypothesis and show that ArsH in solution at room temperature does exist predominantly in the tetrameric form.     

ArsH from the cyanobacterium Synechocystis sp. PCC 6803 is an efficient NADPH-dependent quinone reductase

The cyanobacterium Synechocystis sp. PCC 6803 possesses an arsenic resistance operon that encodes, among others, an ArsH protein. ArsH is a flavin mononucleotide (FMN)-containing protein of unknown function and a member of the family of NADPH-dependent FMN reductases. The nature of its final electron acceptor and the role of ArsH in the resistance to arsenic remained to be clarified. Here we have expressed and purified Synechocystis ArsH and conducted an intensive biochemical study. We present kinetic evidence supporting a quinone reductase activity for ArsH, with a preference for quinones with hydrophobic substituents. By using steady-state activity measurements, as well as stopped-flow and laser-flash photolysis kinetic analyses, it has been possible to establish the mechanism of the process and estimate the values of the kinetic constants. Although the enzyme is able to stabilize the anionic semiquinone form of the FMN, reduction of quinones involves the hydroquinone form of the flavin cofactor, and the enzymatic reaction occurs through a ping-pong-type mechanism. ArsH is able to catalyze one-electron reactions (oxygen and cytocrome c reduction), involving the FMN semiquinone form, but with lower efficiency. In addition, arsH mutants are sensitive to the oxidizing agent menadione, suggesting that ArsH plays a role in the response to oxidative stress caused by arsenite.     

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.     

Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

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.     

Synechocystis DrgA protein functioning as nitroreductase and ferric reductase is capable of catalyzing the Fenton reaction

In order to identify an enzyme capable of Fenton reaction in Synechocystis, we purified an enzyme catalyzing one-electron reduction of t-butyl hydroperoxide in the presence of FAD and Fe(III)-EDTA. The enzyme was a 26 kDa protein, and its N-terminal amino acid sequencing revealed it to be DrgA protein previously reported as quinone reductase [Matsuo M, Endo T and Asada K (1998) Plant Cell Physiol39, 751-755]. The DrgA protein exhibited potent quinone reductase activity and, furthermore, we newly found that it contained FMN and highly catalyzed nitroreductase, flavin reductase and ferric reductase activities. This is the first demonstration of nitroreductase activity of DrgA protein previously identified by a drgA mutant phenotype. DrgA protein strongly catalyzed the Fenton reaction in the presence of synthetic chelate compounds, but did so poorly in the presence of natural chelate compounds. Its ferric reductase activity was observed with both natural and synthetic chelate compounds with a better efficiency with the latter. In addition to small molecular-weight chemical chelators, an iron transporter protein, transferrin, and an iron storage protein, ferritin, turned out to be substrates of the DrgA protein, suggesting it might play a role in iron metabolism under physiological conditions and possibly catalyze the Fenton reaction under hyper-reductive conditions in this microorganism.     

Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+

BACKGROUND: Studies performed within the last decade have indicated that microbial reduction of Fe(III) to Fe(II) is a biologically significant process. The ferric reductase (FeR) from Archaeoglobus fulgidus is the first reported archaeal ferric reductase and it catalyzes the flavin-mediated reduction of ferric iron complexes using NAD(P)H as the electron donor. Based on its catalytic activity, the A. fulgidus FeR resembles the bacterial and eukaryotic assimilatory type of ferric reductases. However, the high cellular abundance of the A. fulgidus FeR (approximately 0.75% of the total soluble protein) suggests a catabolic role for this enzyme as the terminal electron acceptor in a ferric iron-based respiratory pathway [1]. RESULTS: The crystal structure of recombinant A. fulgidus FeR containing a bound FMN has been solved at 1.5 A resolution by multiple isomorphous replacement/ anomalous diffraction (MIRAS) phasing methods, and the NADP+- bound complex of FeR was subsequently determined at 1.65 A resolution. FeR consists of a dimer of two identical subunits, although only one subunit has been observed to bind the redox cofactors. Each subunit is organized around a six-stranded antiparallel beta barrel that is homologous to the FMN binding protein from Desulfovibrio vulgaris. This fold has been shown to be related to a circularly permuted version of the flavin binding domain of the ferredoxin reductase superfamily. The A. fulgidus ferric reductase is further distinguished from the ferredoxin reductase superfamily by the absence of a Rossmann fold domain that is used to bind the NAD(P)H. Instead, FeR uses its single domain to provide both the flavin and the NAD(P)H binding sites. Potential binding sites for ferric iron complexes are identified near the cofactor binding sites. CONCLUSIONS: The work described here details the structures of the enzyme-FMN, enzyme-FMN-NADP+, and possibly the enzyme-FMN-iron intermediates that are present during the reaction mechanism. This structural information helps identify roles for specific residues during the reduction of ferric iron complexes by the A. fulgidus FeR.     

The flavin reductase ActVB from Streptomyces coelicolor: characterization of the electron transferase activity of the flavoprotein form

The flavin reductase ActVB is involved in the last step of actinorhodin biosynthesis in Streptomyces coelicolor. Although ActVB can be isolated with some FMN bound, this form was not involved in the flavin reductase activity. By studying the ferric reductase activity of ActVB, we show that its FMN-bound form exhibits a proper enzymatic activity of reduction of iron complexes by NADH. This shows that ActVB active site exhibits a dual property with regard to the FMN. It can use it as a substrate that goes in and off the active site or as a cofactor to provide an electron transferase activity to the polypeptide.     

Arabinan degrading enzymes from Aspergillus nidulans: Induction and purification

Abstract The expression and distribution of ferric reductase activity was examined in Shewanella putrefaciens MR-1. Formate-dependent ferric reductase was not detected in aerobically grown cells but was readily detectable in anaerobically grown cells. Ferric reductase activity was found exclusively in the membrane fractions, with 54–56% in the outer membrane. In contrast, the majority of formate dehydrogenase was in the soluble fraction with lesser amounts associated with the various membrane fractions. Outer membrane ferric reductase activity was markedly inhibited by p -chloromercuriphenylsulfonate, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, but was unaffected by the presence of alternate electron acceptors (nitrate, nitrite, fumarate, and trimethylamine N -oxide). Both formate and NADH served as electron donors for ferric reductase; activity with l -lactate or NADPH was poor. The addition of FMN markedly stimulated formate- and NADH-dependent ferric reductase.     

Relationship between changes in properties and contents of riboflavin derivatives of NADPH-cytochrome P-450 reductase in the liver microsomes of riboflavin-deficient rats

Weanling male rats were fed a riboflavin-deficient diet for 5-8 weeks, and the decrease in NADPH-cytochrome P-450 reductase (FpT) activity in the liver microsomes was compared with the contents of riboflavin derivatives. The decrease of FpT activity for the reduction of cytochrome c was greater than that for the reduction of ferricyanide. The FpT's of riboflavin-deficient and control rats were indistinguishable in the Ouchterlony immunodiffusion test against anti-FpT, and were shown to have the same molecular weight of 78,000 by SDS-polyacrylamide slab gel electrophoresis. However, the purified FpT of the riboflavin-deficient rats contained 14.2, 4.9, and 1.9 nmol of FAD, FMN, and riboflavin per mg of protein, respectively, while that of the control rats contained 10.6 and 9.5 nmol of FAD and FMN per mg of protein, respectively. After riboflavin injection into the riboflavin-deficient rats, NADPH-cytochrome c reductase activity and FMN content of the FpT were restored to the control levels in 36 h, NADPH-ferricyanide reductase activity recovered in 18 h, and riboflavin content diminished in 18 h. On incubation of the purified FpT of the riboflavin-deficient rats with FMN, NADPH-cytochrome c reductase activity and FMN content were restored to those of control rats. These results indicated that a part of FMN in the FpT of the riboflavin-deficient rats was replaced with FAD and riboflavin.     

Ferric iron reduction and iron assimilation in Saccharomyces cerevisiae.

We have used the yeast Saccharomyces cerevisiae as a model organism to study the role of ferric iron reduction in eucaryotic iron uptake. S. cerevisiae is able to utilize ferric chelates as an iron source by reducing the ferric iron to the ferrous form, which is subsequently internalized by the cells. A gene (FRE1) was identified which encodes a protein required for both ferric iron reduction and efficient ferric iron assimilation, thus linking these two activities. The predicted FRE1 protein appears to be a membrane protein and shows homology to the beta-subunit of the human respiratory burst oxidase. These data suggest that FRE1 is a structural component of the ferric reductase. Subcellular fractionation studies showed that the ferric reductase activity of isolated plasma membranes did not reflect the activity of the intact cells, implying that cellular integrity was necessary for function of the major S. cerevisiae ferric reductase. An NADPH-dependent plasma membrane ferric reductase was partially purified from plasma membranes. Preliminary evidence suggests that the cell surface ferric reductase may, in addition to mediating cellular iron uptake, help modulate the intracellular redox potential of the yeast cell.     

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.     

NADPH-cytochrome P-450 reductase. Physical properties and redox behavior in the absence of the FAD moiety

NADPH-cytochrome P-450 reductase contains one molecule each of FMN and FAD. The FAD moiety has been selectively removed, producing the FMN reductase. The FMN reductase is stable and enzymatic activity is reconstituted with either FAD or FMN. FMN remains tightly bound, but can both dissociate from the FMN site and bind to the vacant FAD site. The amount of FMN bound in the FAD site is minimal under specific experimental conditions. There are at least two conformational subpopulations of the FMN reductase; NADP dissociates readily from one but extremely slowly from the other. Rapid dissociation of NADP is regained upon reconstitution with FAD. The one-electron redox state of the FMN reductase is thermodynamically stabilized, though to a lesser degree than in the holoreductase. When two-electron reduced FMN reductase is exposed to oxygen, a stable species with an absorbance peak at 580 nm forms rapidly and quantitatively. This species has been identified by electron paramagnetic resonance spectroscopy as the neutral radical of FMN and is indistinguishable from the air-stable radical of the holoreductase. The redox behavior of the FMN reductase is in agreement with properties proposed previously for the FMN site.     

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.     

The reactions of heme- and verdoheme-heme oxygenase-1 complexes with FMN-depleted NADPH-cytochrome P450 reductase. Electrons required for verdoheme oxidation can be transferred through a pathway not involving FMN

Electrons utilized in the heme oxygenase (HO) reaction are provided by NADPH-cytochrome P450 reductase (CPR). To investigate the electron transfer pathway from CPR to HO, we examined the reactions of heme and verdoheme, the second intermediate in the heme degradation, complexed with rat HO-1 (rHO-1) using a rat FMN-depleted CPR; the FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 m KBr. Degradation of heme in complex with rHO-1 did not occur with FMN-depleted CPR, notwithstanding that the FMN-depleted CPR was able to associate with the heme-rHO-1 complex with a binding affinity comparable with that of the wild-type CPR. Thus, the first electron to reduce the ferric iron of heme complexed with rHO-1 must be transferred from FMN. In contrast, verdoheme was converted to the ferric biliverdin-iron chelate with FMN-depleted CPR, and this conversion was inhibited by ferricyanide, indicating that electrons are certainly required for conversion of verdoheme to a ferric biliverdin-iron chelate and that they can be supplied from the FMN-depleted CPR through a pathway not involving FMN, probably via FAD. This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR. Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine. Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR.     

The effect of insulin and glucose on fructose-2,6-P2 in hepatocytes

When rats were kept on a riboflavin-deficient diet, NADPH-cytochrome c and NADPH-ferricyanide reductase activities of the liver microsomes (deficient microsomes) decreased to 27% and 40% of the corresponding controls. To elucidate the unbalanced decrease of these activities in deficient microsomes, enzymological and immunochemical properties of the NADPH-cytochrome P-450 reductase in the liver microsomes of riboflavin-deficient rats were compared with those of control rats. Judging from quantitative immunoprecipitation, the amount of the reductase protein in the deficient microsomes was 67% of control, whereas the FAD and FMN contents in the immunoprecipitates were 110% and 59% of control, respectively. When the reductase was purified from the deficient microsomes, it contained 18.0 and 10.9 nmoles of FAD and FMN, respectively, per mg of protein, while the control enzyme contained 14.5 and 14.3 nmoles of the flavins, respectively. These and other lines of evidence suggest the existence of an abnormal NADPH-cytochrome P-450 reductase, having unbalanced contents of FAD and FMN, in deficient microsomes.     

The Structural and Functional Basis of Catalysis Mediated by NAD(P)H:acceptor Oxidoreductase (FerB) of Paracoccus denitrificans

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.     

Ferrisiderophore reductases of Pseudomonas. Purification, properties and cellular location of the Pseudomonas aeruginosa ferripyoverdine reductase.

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M(r) 27,000-28,000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical M(r) as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.