The single NqrB and NqrC subunits in the Na+-translocating NADH: Quinone oxidoreductase (Na+-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe–2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na⁺-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na⁺-NQR contains approximately 1.7 mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na⁺-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na⁺-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na⁺-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc₁ and b₆f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na⁺-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na⁺-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na⁺-NQR.     

Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na-translocating NADH:quinone oxidoreductase

The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR), and a 99.5 kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1 mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na⁺-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na⁺-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na⁺-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na⁺-NQR. A new, “electron loop” mechanism is proposed for Na⁺-NQR, involving an electroneutral Na⁺/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The Conformational Changes Induced by Ubiquinone Binding in the Na+-pumping NADH:Ubiquinone Oxidoreductase (Na+-NQR) Are Kinetically Controlled by Conserved Glycines 140 and 141 of the NqrB Subunit

Na⁺-pumping NADH:ubiquinone oxidoreductase (Na⁺-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na⁺-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na⁺-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.     

Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications

The recently determined crystal structure of NhaA, the Na ⁺/H ⁺ antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.     

Sodium-translocating NADH:quinone oxidoreductase as a redox-driven ion pump

The Na⁺-translocating NADH:ubiquinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria. This enzyme is an analogous but not homologous counterpart of mitochondrial Complex I. Na⁺-NQR drives the same chemistry and also uses released energy to translocate ions across the membrane, but it pumps Na⁺ instead of H⁺. Most likely the mechanism of sodium pumping is quite different from that of proton pumping (for example, it could not accommodate the Grotthuss mechanism of ion movement); this is why the enzyme structure, subunits and prosthetic groups are completely special. This review summarizes modern knowledge on the structural and catalytic properties of bacterial Na⁺-translocating NADH:quinone oxidoreductases. The sequence of electron transfer through the enzyme cofactors and thermodynamic properties of those cofactors is discussed. The resolution of the intermediates of the catalytic cycle and localization of sodium-dependent steps are combined in a possible molecular mechanism of sodium transfer by the enzyme.     

Cys377 residue in NqrF subunit confers Ag<Superscript>+</Superscript> sensitivity of Na<Superscript>+</Superscript>-translocating NADH:quinone oxidoreductase from <Emphasis Type="Italic">Vibrio harveyi</Emphasis>

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a component of the respiratory chain of various bacteria that generates a redox-driven transmembrane electrochemical Na⁺ potential. The Na⁺-NQR activity is known to be specifically inhibited by low concentrations of silver ions. Replacement of the conserved Cys377 residue with alanine in the NqrF subunit of Na⁺-NQR from Vibrio harveyi resulted in resistance of the enzyme to Ag⁺ and to other heavy metal ions. Analysis of the catalytic activity also showed that the rate of electron input into the mutant Na⁺-NQR decreased by about 14-fold in comparison to the wild type enzyme, whereas all other properties of _NqrFC377A Na⁺-NQR including its stability remained unaffected.     

Continuous fluorescence-based measurement of redox-driven sodium ion translocation

Investigation of the mechanism of sodium ion pumping enzymes requires methods to follow the translocation of sodium ions by the purified and reconstituted proteins in vitro. Here, we describe a protocol that allows following the accumulation of Na⁺ in proteoliposomes by the Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae using the sodium-sensitive fluorophor sodium green. In the presence of a regenerative system for its substrate NADH, the Na⁺-NQR accumulates Na⁺ in the proteoliposomes which is visible as a change in fluorescence.     

The single NqrB and NqrC subunits in the Na(+)-translocating NADH: Quinone oxidoreductase (Na(+)-NQR) from Vibrio cholerae each carry one covalently attached FMN

The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is the prototype of a novel class of flavoproteins carrying a riboflavin phosphate bound to serine or threonine by a phosphodiester bond to the ribityl side chain. This membrane-bound, respiratory complex also contains one non-covalently bound FAD, one non-covalently bound riboflavin, ubiquinone-8 and a [2Fe-2S] cluster. Here, we report the quantitative analysis of the full set of flavin cofactors in the Na(+)-NQR and characterize the mode of linkage of the riboflavin phosphate to the membrane-bound NqrB and NqrC subunits. Release of the flavin by β-elimination and analysis of the cofactor demonstrates that the phosphate group is attached at the 5'-position of the ribityl as in authentic FMN and that the Na(+)-NQR contains approximately 1.7mol covalently bound FMN per mol non-covalently bound FAD. Therefore, each of the single NqrB and NqrC subunits in the Na(+)-NQR carries a single FMN. Elimination of the phosphodiester bond yields a dehydro-2-aminobutyrate residue, which is modified with β-mercaptoethanol by Michael addition. Proteolytic digestion followed by mass determination of peptide fragments reveals exclusive modification of threonine residues, which carry FMN in the native enzyme. The described reactions allow quantification and localization of the covalently attached FMNs in the Na(+)-NQR and in related proteins belonging to the Rhodobacter nitrogen fixation (RNF) family of enzymes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.     

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB–NqrD subunit interface

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is the major Na⁺ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na⁺-NQR. While the mechanisms underlying Na⁺-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na⁺-NQR. An in silico model of the NqrB–D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na⁺-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB–D interface underwent a non-physiological transition after 350 ns.

     

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na⁺ across the bacterial membrane. The Na⁺-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na⁺-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na⁺-NQR is discussed.     

A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy-dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino-terminal end of the transporter were fused to the Escherichia coli enzymes aikaline phosphatase (PhoA) or β-galactosidase (LacZ). The enzymatic activity of 16 HoxN-PhoA and 15 HoxN-LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two-dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.     

Catalytic properties of the isolated diaphorase fragment of the NAD-reducing [NiFe]-hydrogenase from Ralstonia eutropha

The NAD⁺-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H₂-driven reduction of NAD⁺, as well as reverse electron transfer from NADH to H⁺, in the presence of O₂. It comprises six subunits, HoxHYFUI₂, and incorporates a [NiFe] H⁺/H₂ cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD⁺ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD⁺/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD⁺, respectively. Catalysis in both directions is product inhibited with K _I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O₂, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis.     

Membrane topology of the pBR322 tetracycline resistance protein. TetA-PhoA gene fusions and implications for the mechanism of TetA membrane insertion.

The tetracycline resistance gene of pBR322 encodes a 41-kDa inner membrane protein (TetA) that acts as a tetracycline/H+ antiporter. Based on hydrophobicity profiles, we identified 12 potential transmembrane segments in TetA. We used oligonucleotide deletion mutagenesis to fuse alkaline phosphatase (PhoA) to the C-terminal edge of each of the predicted periplasmic and cytoplasmic segments of TetA. In general, the PhoA activities of the TetA-PhoA fusions support a TetA topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, several TetA-PhoA fusions have unexpected properties. One PhoA fusion to a predicted cytoplasmic segment (C6) has high activity. However, previous protease accessibility studies on the related Tn10 TetA protein indicated that C6 is cytoplasmically localized as predicted (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). PhoA fusions to three predicted periplasmic segments (P1, P2, and P5) have low to intermediate activity. In each case, the preceding transmembrane segment (TM1, TM3, and TM9) contains an aspartate (Asp17, Asp86, and Asp287). We show that these aspartates act like signal sequence mutations for PhoA export: (i) Asp----Ala mutations increase the PhoA activity of fusions to P1, P2, and P5. (ii) The signal sequence mutation suppressor prlA402 increases the PhoA activity of these same fusions. We also show that the aspartates in TM1, TM3, and TM9 are critical for wild-type TetA function; they are conserved in related TetA proteins and Asp----Ala mutations reduce or eliminate tetracycline resistance. The properties of the anomalous TetA-PhoA fusions suggest that TetA sequences C-terminal to some cytoplasmic and periplasmic segments are required for the proper localization of those segments, i.e. long range interactions may be more important in determining the membrane topology of TetA than suggested in some general models.     

The sodium pumping NADH:quinone oxidoreductase (Na+-NQR), a unique redox-driven ion pump

The Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique Na⁺ pumping respiratory complex found only in prokaryotes, that plays a key role in the metabolism of marine and pathogenic bacteria, including Vibrio cholerae and other human pathogens. Na⁺-NQR is the main entrance for reducing equivalents into the respiratory chain of these bacteria, catalyzing the oxidation of NADH and the reduction of quinone, the free energy of this redox reaction drives the selective translocation of Na⁺ across the cell membrane, which energizes key cellular processes. In this review we summarize the unique properties of Na⁺-NQR in terms of its redox cofactor composition, electron transfer reactions and a possible mechanism of coupling and pumping.     

The physiological role of riboflavin transporter and involvement of FMN-riboswitch in its gene expression in Corynebacterium glutamicum

Riboflavin is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which work as cofactors of numerous enzymes. Understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. In this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expression of riboflavin transporter in Corynebacterium glutamicum. Disruption of the ribA gene that encodes bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase in C. glutamicum suppressed growth in the absence of supplemental riboflavin. The growth was fully recovered upon supplementation with 1 μM riboflavin, albeit at reduced intracellular concentrations of FMN and FAD during the log phase. Concomitant disruption of the ribA and ribM gene that encodes a riboflavin transporter exacerbated supplemental riboflavin requirement from 1 μM to 50 μM. RibM expression in FMN-rich cells was about 100-fold lower than that in FMN-limited cells. Mutations in putative FMN-riboswitch present immediately upstream of the ribM gene abolished the FMN response. This 5′UTR sequence of ribM constitutes a functional FMN-riboswitch in C. glutamicum.     

The membrane topology of the Rhizobium meliloti C4-dicarboxylate permease (DctA) as derived from protein fusions with Escherichia coli K12 alkaline phosphatase (PhoA) and β-galactosidase (LacZ)

The Rhizobium meliloti dctA gene encodes the C₄-dicarboxylate permease which mediates uptake of C₄-dicarboxylates, both in free-living and symbiotic cells. Based on the hydrophobicity of the DctA protein, 12 putative membrane spanning regions were predicted. The membrane topology was further analysed by isolating in vivo fusions of DctA to Escherichia coli alkaline phosphatase (PhoA) and E. coli β-galactosidase (LacZ). Of 10 different fusions 7 indicated a periplasmic and 3 a cytoplasmic location of the corresponding region of the DctA protein. From these data a two-dimensional model of DctA was constructed which comprised twelve transmembrane α-helices with the amino-terminus and the carboxy-terminus located in the cytoplasm. In addition, four conserved amino acid motifs present in many eukaryotic and prokaryotic transport proteins were observed.     

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.