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).     

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.     

FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus

The Na(+)-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue. Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits. From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit. The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.     

Recent progress in the Na(+)-translocating NADH-quinone reductase from the marine Vibrio alginolyticus

The respiratory chain of Gram-negative marine and halophilic bacteria has a Na(+)-dependent NADH-quinone reductase that functions as a primary Na(+) pump. The Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is composed of six structural genes (nqrA to nqrF). The NqrF subunit has non-covalently bound FAD. There are conflicting results on the existence of other flavin cofactors. Recent studies revealed that the NqrB and NqrC subunits have a covalently bound flavin, possibly FMN, which is attached to a specified threonine residue. A novel antibiotic, korormicin, was found to specifically inhibit the NQR complex. From the homology search of the nqr operon, it was found that the Na(+)-pumping NQR complex is widely distributed among Gram-negative pathogenic bacteria.     

Sodium-Dependent Movement of Covalently Bound FMN Residue(s) in Na(+)-Translocating NADH:Quinone Oxidoreductase

Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is a component of respiratory electron-transport chain of various bacteria generating redox-driven transmembrane electrochemical Na(+) potential. We found that the change in Na(+) concentration in the reaction medium has no effect on the thermodynamic properties of prosthetic groups of Na(+)-NQR from Vibrio harveyi, as was revealed by the anaerobic equilibrium redox titration of the enzyme's EPR spectra. On the other hand, the change in Na(+) concentration strongly alters the EPR spectral properties of the radical pair formed by the two anionic semiquinones of FMN residues bound to the NqrB and NqrC subunits (FMN(NqrB) and FMN(NqrC)). Using data obtained by pulse X- and Q-band EPR as well as by pulse ENDOR and ELDOR spectroscopy, the interspin distance between FMN(NqrB) and FMN(NqrC) was found to be 15.3 ? in the absence and 20.4 ? in the presence of Na(+), respectively. Thus, the distance between the covalently bound FMN residues can vary by about 5 ? upon changes in Na(+) concentration. Using these results, we propose a scheme of the sodium potential generation by Na(+)-NQR based on the redox- and sodium-dependent conformational changes in the enzyme.     

Structural and Functional Investigation of Flavin Binding Center of the NqrC Subunit of Sodium-Translocating NADH:Quinone Oxidoreductase from Vibrio harveyi

Na⁺-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium.     

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.     

Membrane topology mapping of the Na+-pumping NADH: quinone oxidoreductase from Vibrio cholerae by PhoA-green fluorescent protein fusion analysis

The membrane topologies of the six subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR) from Vibrio cholerae were determined by a combination of topology prediction algorithms and the construction of C-terminal fusions. Fusion expression vectors contained either bacterial alkaline phosphatase (phoA) or green fluorescent protein (gfp) genes as reporters of periplasmic and cytoplasmic localization, respectively. A majority of the topology prediction algorithms did not predict any transmembrane helices for NqrA. A lack of PhoA activity when fused to the C terminus of NqrA and the observed fluorescence of the green fluorescent protein C-terminal fusion confirm that this subunit is localized to the cytoplasmic side of the membrane. Analysis of four PhoA fusions for NqrB indicates that this subunit has nine transmembrane helices and that residue T236, the binding site for flavin mononucleotide (FMN), resides in the cytoplasm. Three fusions confirm that the topology of NqrC consists of two transmembrane helices with the FMN binding site at residue T225 on the cytoplasmic side. Fusion analysis of NqrD and NqrE showed almost mirror image topologies, each consisting of six transmembrane helices; the results for NqrD and NqrE are consistent with the topologies of Escherichia coli homologs YdgQ and YdgL, respectively. The NADH, flavin adenine dinucleotide, and Fe-S center binding sites of NqrF were localized to the cytoplasm. The determination of the topologies of the subunits of Na⁺-NQR provides valuable insights into the location of cofactors and identifies targets for mutagenesis to characterize this enzyme in more detail. The finding that all the redox cofactors are localized to the cytoplasmic side of the membrane is discussed.     

Covalent binding of flavins to RnfG and RnfD in the Rnf complex from Vibrio cholerae

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na+-NQR complexes.     

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.

     

Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy. Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a [2Fe-2S] cluster. An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration. The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions. The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species. Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium. Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM. This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme. These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.     

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.     

The side chain of ubiquinone plays a critical role in Na+ translocation by the NADH-ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae

The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is an essential bacterial respiratory enzyme that generates a Na⁺ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na⁺ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na⁺ translocation using a series of synthetic UQs with Vibrio cholerae Na⁺-NQR reconstituted into liposomes. UQ₀ that has no side chain and UQ_CH3 and UQ_C2H5, which have methyl and ethyl side chains, respectively, were catalytically reduced by Na⁺-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na⁺ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na⁺ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C₃H₇) or longer significantly restored Na⁺ translocation. It has been considered that Na⁺ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na⁺ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na⁺ translocation.     

Riboflavin is an active redox cofactor in the Na+-pumping NADH: quinone oxidoreductase (Na+-NQR) from Vibrio cholerae

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR.     

FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.     

The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities

The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant human liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine. Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for riboflavin, MAO A and MAO B were expressed separately under control of a galactose inducible GAL10/CYC1 promoter in the presence of N(10)-omega-hydroxypentyl-isoalloxazine as the only available riboflavin analogue. Analysis of mitochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin. Catalytic activities, as monitored by kynuramine oxidation, are equivalent to (MAO A) or 2-fold greater (MAO B) than control preparations expressed in the presence of riboflavin. Although N(10)-omega-hydroxypentyl-isoalloxazine is unable to support growth of riboflavin auxotrophic S. cerevisiae, it is converted to the FMN level by yeast cell free extracts. The FMN form of the analogue is converted to the FAD level by the yeast FAD synthetase, as shown by expression of the recombinant enzyme in Escherichia coli. These data show that the ribityl hydroxyl groups of the FAD moiety are not required for covalent flavinylation or catalytic activities of monoamine oxidases A and B. This is in contrast to the suggestion based on mutagenesis studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylation reaction of MAO B (Zhou et al., J. Biol. Chem. 273 (1998) 14862-14868).     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.     

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD⁺, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.