Identity of the subunits and the stoicheiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase.

Trimethylamine dehydrogenases from bacterium W3A1 and Hyphomicrobium X and the dimethylamine dehydrogenase from Hyphomicrobium X were found to contain only one kind of subunit. The millimolar absorption coefficient of a single [4Fe-4S] cluster in trimethylamine dehydrogenase from bacterium W3A1 was estimated to be 14.8 mM-1 . cm-1 at 443 nm. From this value a 1:1 stoicheiometry of the prosthetic groups, 6-S-cysteinyl-FMN and the [4Fe-4S] cluster, was established. Millimolar absorption coefficients of the three enzymes were in the range 49.4-58.7 mM-1 . cm-1 at approx. 440 nm. This range of values is consistent with the presence of two [4Fe-4S] clusters and two flavin residues, for which the millimolar absorption coefficient had earlier been found to be 12.3 mM-1 . cm-1 at 437 nm. The N-terminal amino acid was alanine in each of the three enzymes. Sequence analysis of the first 15 residues from the N-terminus of dimethylamine dehydrogenase indicated a single unique sequence. Two identical subunits, each containing covalently bound 6-S-cysteinyl-FMN and a [4Fe-4S] cluster, in each of the enzymes are therefore indicated.     

Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 1. The influence of substrate binding to reduced trimethylamine dehydrogenase on the intramolecular electron transfer between its prosthetic groups.

The trimethylamine dehydrogenase of bacterium W3A1 is reduced with the formation of a triplet state in which two electrons, derived from the substrate, are distributed between the [4Fe-4S] cluster and 6-S-cysteinyl-FMN semiquinone. In titration experiments at pH 8.5 about 1.0 mol of dimethylamine or 0.5 mol of trimethylamine per mol of the enzyme is required to titrate the enzyme to an endpoint. At pH values less than 8.0, however, an excess of trimethylamine is required to obtain maximal yield of the g = 4 e.p.r. signal, characteristic of the triplet state, or maximal absorbance at 365 nm which indicates formation of the flavin semiquinone. The binding of 0.86 mol of trimethylamine per mol of the enzyme could be detected by a gel chromatographic method. When the enzyme is titrated with dithionite in the presence of tetramethylammonium chloride, an endpoint is reached after the uptake of two electrons which give rise to the triplet state, whereas three electrons are consumed in the absence of tetramethylammonium chloride to reduce the enzyme completely. The enzyme is inhibited noncompetitively by tetramethylammonium chloride and the slopes of double reciprocal plots are a concave upwards function of inhibitor concentration. The data indicate the presence of a binding site for the substrate and other amines on the reduced enzyme which enhances the proportion of enzyme in the triplet state.     

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre.

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.     

Coenzyme M binds to a [4Fe-4S] cluster in the active site of heterodisulfide reductase as deduced from EPR studies with the [33S]coenzyme M-treated enzyme

Heterodisulfide reductase (Hdr) from methanogenic Archaea catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Upon reaction of the oxidized enzyme with CoM-SH a unique paramagnetic species is formed, which has been shown to be due to a novel type of [4Fe-4S](3+) cluster. In this work, it was addressed whether CoM-SH is directly attached to this [4Fe-4S] cluster using CoM-(33)SH as substrate and purified Hdr from Methanothermobacter marburgensis and Methanosarcina barkeri. With both enzymes treatment with CoM-(33)SH in the presence of duroquinone as an oxidant resulted in a significant broadening of the electron paramagnetic resonance spectrum as compared to CoM-SH as substrate. The signal broadening resulted from an unresolved anisotropic hyperfine coupling between the (33)S nucleus and the paramagnetic center. The results provide compelling evidence for a direct binding of CoM-SH to the [4Fe-4S] cluster in the active site of the enzyme.     

The flavoprotein subcomplex of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria: insights into the mechanisms of NADH oxidation and NAD+ reduction from protein film voltammetry

Complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria contains 45 different subunits and nine redox cofactors. NADH is oxidized by a noncovalently bound flavin mononucleotide (FMN), then seven iron-sulfur clusters transfer the two electrons to quinone, and four protons are pumped across the inner mitochondrial membrane. Here, we use protein film voltammetry to investigate the mechanisms of NADH oxidation and NAD+ reduction in the simplest catalytically active subcomplex of complex I, the flavoprotein (Fp) subcomplex. The Fp subcomplex was prepared using chromatography and contained the 51 and 24 kDa subunits, the FMN, one [4Fe-4S] cluster, and one [2Fe-2S] cluster. The reduction potential of the FMN in the enzyme's active site is lower than that of free FMN (thus, the oxidized state of the FMN is most strongly bound) and close to the reduction potential of NAD+. Consequently, the catalytic transformation is reversible. Electrocatalytic NADH oxidation by subcomplex Fp can be explained by a model comprising substrate mass transport, the Michaelis-Menten equation, and interfacial electron transfer kinetics. The difference between the "catalytic" potential and the FMN potential suggests that the flavin is reoxidized before NAD+ is released or that intramolecular electron transfer from the flavin to the [4Fe-4S] cluster influences the catalytic rate. NAD+ reduction displays a marked activity maximum, below which the catalytic rate decreases sharply as the driving force increases. Two possible models reproduce the observed catalytic waveshapes: one describing an effect from reducing the proximal [2Fe-2S] cluster and the other the enhanced catalytic ability of the semiflavin state.     

The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases.

Pig liver electron transfer flavoprotein (ETF) is rapidly reduced by sarcosine and dimethylglycine dehydrogenases to the anionic semiquinone form, the subsequent formation of the flavoquinol form being a much slower process. In the presence of tetrahydrofolate the yield of anionic semiquinone at the end of the rapid phase of reduction of ETF is only about 10% less than without tetrahydrofolate, as judged by e.p.r. spectroscopy. Tetrahydrofolate does not alter the rate of reduction of ETF by either sarcosine or dimethylglycine dehydrogenase. Nevertheless, it was clearly demonstrated that tetrahydrofolate is a substrate for both sarcosine and dimethylglycine dehydrogenases and is converted to N5,10-methylenetetrahydrofolate.     

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S]3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX31-39CCX35-36CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (gzyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel [4Fe-4S] cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with an S3(O/N)1 geometry in HdrB and the HDR holoenzyme. The N-terminal CCG domain is suggested to provide ligands to the Zn site.     

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties.In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH?8 by 230?mV to 400?mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.     

Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe-4S] cluster with one non-cysteine ligand.

Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one [3Fe-4S]1+,0 and one [4Fe-4S]2+,1+ core cluster when aerobically isolated. The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters. Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-'edge' electrode promoted by neomycin shows that, when reduced, the [3Fe-4S]0 centre reacts rapidly with Fe(II) ion to form a [4Fe-4S]2+ cluster. The latter, which can be reduced at a redox potential similar to that of the other [4Fe-4S] cluster, must include non-thiolate ligation. We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin. The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d. and e.p.r. spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state. This cluster provides a plausible model for the ligation states of the [4Fe-4S]1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase.     

Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies.

Preparations of nitrate reductase in the resting state from Pseudomonas aeruginosa exhibit an Mo(V) e.p.r. signal. Progressive reduction of the enzyme results at first in the intensification and then in the disappearance of the signal. Three different species of Mo(V) were detected by e.p.r. These are the high-pH species (g1 = 1.9871; g2 = 1.9795; g3 = 1.9632) and nitrate and nitrite complexes of a low-pH species (respectively g1 = 2.0004; g2 = 1.9858; g3 = 1.9670; and g1 = 1.9975; g2 = 1.9848; g3 = 1.9652). These signals are closely analogous to those for the enzyme from Escherichia coli described by Vincent & Bray [(1978) Biochem. J. 171, 639-647]. Signals typical of iron-sulphur clusters were also detected. In the oxidized enzyme these are believed to arise from a [3Fe-4S] cluster (g = 2.01) and in the reduced enzyme from an unusual low-potential [4Fe-4S]+ cluster (g1 = 2.054; g2 = 1.952; g3 = 1.878). The iron-sulphur centres were also studied in a 'high-catalytic-activity' form of the enzyme. Reduction with Na2S2O4 resulted in the formation of a complex signal with g values at 2.054, 1.952, 1.928, 1.903 and 1.878. The signal could be deconvoluted by reductive titration of the enzyme into two species (g1 = 2.054; g2 = 1.952; g3 = 1.878; and g1 = 2.036; g2 = 1.928; g3 = 1.903). The degradation of a [4Fe-4S] into a [3Fe-4S] cluster in the enzyme is suggested by these studies, the process being dependent on the method used to purify the enzyme. The addition of nitrate to the reduced enzyme results in the oxidation of Mo(IV) to Mo(V) and of all the iron-sulphur centres.     

On the ATP-Dependent Activation of the Radical Enzyme (R)-2-Hydroxyisocaproyl-CoA Dehydratase

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 ? resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.     

Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase

We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV [4Fe-4S] cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a [3Fe-4S] cluster in DmsA, suggesting that the [3Fe-4S] cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized [3Fe-4S] cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered [3Fe-4S] cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized [3Fe-4S] cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD of DmsA.     

In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.     

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.     

Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa.

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.     

Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling.

The localization of prominent proteins in intact cells of two methylotrophic bacteria, Hyphomicrobium sp. strain X and bacterium W3A1, was investigated by radiochemical labeling with [14C]isethionyl acetimidate. In bacterium W3A1, trimethylamine dehydrogenase was not labeled by the reagent and is, therefore, an intracellular protein, whereas the periplasmic location of the methylamine and methanol dehydrogenases was evidenced by being readily labeled in intact cells. Similarly, an intracellular location of the trimethylamine and dimethylamine dehydrogenases in Hyphomicrobium sp. strain X was indicated, whereas methanol dehydrogenase was periplasmic.     

Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster in PsaC of photosystem I. The photoreduction of FA is independent of FB.

The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups

The membrane-bound alcohol dehydrogenase of Gluconacetobacter diazotrophicus contains one pyrroloquinoline quinone moiety (PQQ), one [2Fe-2S] cluster, and four c-type cytochromes. Here, we describe a novel and inactive enzyme. ADHi, similarly to ADHa, is a heterodimer of 72- and 44-kDa subunits and contains the expected prosthetic groups. However, ADHa showed a threefold molecular mass as compared to ADHi. Noteworthy, the PQQ, the [2Fe-2S] and most of the cytochromes in purified ADHi is in the oxidized form, contrasting with ADHa where the PQQ-semiquinone is detected and the [2Fe-2S] cluster as well as the cytochromes c remained fully reduced after purification. Reduction kinetics of the ferricyanide-oxidized enzymes showed that while ADHa was brought back by ethanol to its full reduction state, in ADHi, only one-quarter of the total heme c was reduced. The dithionite-reduced ADHi was largely oxidized by ubiquinone-2, thus indicating that intramolecular electron transfer is not impaired in ADHi. The acidic pH of the medium might be deleterious for the membrane-bound ADH by causing conformational changes leading to changes in the relative orientation of heme groups and shift of corresponding redox potential to higher values. This would hamper electron transfer resulting in the low activity observed in ADHi.     

Histamine dehydrogenase from Rhizobium sp.: gene cloning, expression in Escherichia coli, characterization and application to histamine determination

The gene encoding histamine dehydrogenase in Rhizobium sp. 4--9 has been cloned and overexpressed in Escherichia coli. The coding region of the gene was 2,079 bp and encoded a protein of 693 amino acids with a calculated molecular mass of 76,732 Da. This histamine dehydrogenase was related to histamine dehydrogenase from Nocardioides simplex (54.5% identical), trimethylamine dehydrogenase from Methylophilus methylotrophus (39.3% identical) and dimethylamine dehydrogenase from Hyphomicrobium X (38.1% identical), which have a covalent 6-S-cysteinyl flavin mononucleotide and a [4Fe--4S] cluster as redox cofactors. Sequence alignment and a UV-visible absorption spectrum supported the presence of these cofactors in this histamine dehydrogenase. The investigation of the enzymatic properties suggested that this enzyme exhibited the most excellent substrate specificity toward histamine among all amine oxidases or dehydrogenases found to date. The recombinant enzyme was able to be used for the colorimetric determination of histamine, which gave a linear calibration curve and identical data with conventional methods.