EPR signals assigned to Fe/S cluster N1c of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) derive from cluster N1a

The proton-pumping NADH:ubiquinone oxidoreductase, which is also called respiratory complex I, transfers electrons from NADH to ubiquinone via one flavin mononucleotide (FMN) and up to nine iron-sulfur clusters. A structural minimal form of complex I consisting of 14 different subunits called NuoA to NuoN (or Nqo1 to Nqo14) is found in bacteria. The isolated Escherichia coli complex I can be split into a NADH dehydrogenase fragment, a connecting fragment, and a membrane fragment. The soluble NADH dehydrogenase fragment represents the electron input part of the complex and consists of the subunits NuoE, F, and G. The FMN and four iron-sulfur clusters have been detected in this fragment by means of EPR spectroscopy. One of the EPR signals, called N1c, has spectral properties, which are not found in preparations of the complex from other organisms. Therefore, it is attributed to an additional binding motif on NuoG, which is present only in a few bacteria including E. coli. Here, we show by means of EPR spectroscopic analysis of the NADH dehydrogenase fragment containing site-directed mutations on NuoG that the EPR signals in question derived from cluster N1a on NuoE. The mutations in NuoG disturbed the assembly of the overproduced NADH dehydrogenase fragment indicating that a yet undetected cluster might be bound to the additional motif. Thus, there is no third binuclear iron-sulfur "N1c" in the E. coli complex I but an additional tetranuclear cluster that may be coined N7.     

Substrate-induced conformational change in bacterial complex I

The mechanism coupling electron transfer and proton pumping in respiratory complex I (NADH-ubiquinone oxidoreductase) has not been established, but it has been suggested that it involves conformational changes. Here, the influence of substrates on the conformation of purified complex I from Escherichia coli was studied by cross-linking and electron microscopy. When a zero-length cross-linking reagent was used, the presence of NAD(P)H, in contrast to that of NAD+, prevented the formation of cross-links between the hydrophilic subunits of the complex, including NuoB, NuoI, and NuoCD. Comparisons using different cross-linkers suggested that NuoB, which is likely to coordinate the key iron-sulfur cluster N2, is the most mobile subunit. The presence of NAD(P)H led also to enhanced proteolysis of subunit NuoG. These data indicate that upon NAD(P)H binding, the peripheral arm of the complex adopts a more open conformation, with increased distances between subunits. Single particle analysis showed the nature of this conformational change. The enzyme retains its L-shape in the presence of NADH, but exhibits a significantly more open or expanded structure both in the peripheral arm and, unexpectedly, in the membrane domain also.     

Transport of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> by an antiporter-related subunit from the <Emphasis Type="Italic">Escherichia coli </Emphasis>NADH dehydrogenase I produced in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na⁺ rather than H⁺. To elucidate the mechanism of Na⁺ transport, the C-terminally truncated NuoL subunit (NuoL_N) which is related to Na⁺/H⁺ antiporters was expressed as a protein A fusion protein (ProtA–NuoL_N) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA–NuoL_N catalyzed the uptake of Na⁺ and K⁺ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA–NuoL_N translocated Na⁺ and K⁺ independently from other complex I subunits. Na⁺ transport by ProtA–NuoL_N was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na⁺/H⁺ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Amino acid residues associated with cluster N3 in the NuoF subunit of the proton-translocating NADH-quinone oxidoreductase from Escherichia coli

The NuoF subunit, which harbors NADH-binding site, of Escherichia coli NADH-quinone oxidoreductase (NDH-1) contains five conserved cysteine residues, four of which are predicted to ligate cluster N3. To determine this coordination, we overexpressed and purified the NuoF subunit and NuoF+E subcomplex in E. coli. We detected two distinct EPR spectra, arising from a [4Fe-4S] cluster (g(x,y,z)=1.90, 1.95, and 2.05) in NuoF, and a [2Fe-2S] cluster (g(x,y,z)=1.92, 1.95, and 2.01) in NuoE subunit. These clusters were assigned to clusters N3 and N1a, respectively. Based on the site-directed mutagenesis experiments, we identified that cluster N3 is ligated to the 351Cx2Cx2Cx40C398 motif.     

Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata

Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe²⁺ and 8 S^2?; ferredoxin II has 4 Fe²⁺ and 4 S^2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O₂, O^?₂, H₂O₂ and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C₂H₂ reduction and H₂ evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO₂ and light or in the presence of glutamate in darkness under anaerobic conditions.     

The Origin of Cluster N2 of the Energy-Transducing NADH–Quinone Oxidoreductase: Comparisons of Phylogenetically Related Enzymes

NADH–quinone (Q) oxidoreductase is a large and complex redox proton pump, which utilizes the free energy derived from oxidation of NADH with lipophilic electron/proton carrier Q to translocate protons across the membrane to generate an electrochemical proton gradient ( ). Although its molecular mechanism is largely unknown, recent biochemical, biophysical, and molecular biological studies have revealed that particular subunits and cofactors play an essential role in the energy-coupling reaction. Based on these latest experimental data, we exhaustively analyzed the sequence information available from evolutionarily related enzymes such as [NiFe] hydrogenases. We found significant and conserved sequence differences in the PSST/Nqo6/NuoB, 49kDa/Nqo4/NuoD, and ND1/Nqo8/NuoH subunit homologs between complex I/NDH-1 and [NiFe] hydrogenases. The alterations, especially in the postulated ligand motif for cluster N2 in the PSST/Nqo6/NuoB subunits, appear to be evolutionarily important in determining the physiological function of complex I/NDH-1. These observations led us to propose a hypothetical evolutionary scheme: during the course of evolution, drastic changes have occurred in the putative cluster N2 binding site in the PSST/Nqo6/NuoB subunit and the progenitors of complex I/NDH-1 have concurrently become to utilize a lipophilic electron/proton carrier such as Q as its physiological substrate. This scheme provides new insights into the structure and function relationship of complex I/NDH-1 and may help us understand its energy-coupling mechanism.     

The coupling mechanism of respiratory complex I — A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

The coupling mechanism of respiratory complex I - A structural and evolutionary perspective

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Genetic Analysis of the nuo Locus,Which Encodes the Proton-Translocating NADH Dehydrogenase in Escherichia coli

Complex I (EC 1.6.99.3) of the bacterium Escherichia coli is considered to be the minimal form of the type I NADH dehydrogenase, the first enzyme complex in the respiratory chain. Because of its small size and relative simplicity, the E. coli enzyme has become a model used to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex. To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus, which encodes the 14 Nuo subunits comprising E. coli complex I. Here we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.

Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.99.3), a type I NADH dehydrogenase that couples the oxidation of NADH to the generation of a proton motive force, is the first enzyme complex of the respiratory chain (2, 35, 47). The Escherichia coli enzyme, considered to be the minimal form of complex I, consists of 14 subunits instead of the 40 to 50 subunits associated with the homologous eukaryotic mitochondrial enzyme (17, 29, 30, 48–50). E. coli also possesses a second NADH dehydrogenase, NDH-II, which does not generate a proton motive force (31). E. coli complex I resembles eukaryotic complex I in many ways (16, 17, 30, 49): it performs the same enzymatic reaction and is sensitive to a number of the same inhibitors, it consists of subunits homologous to those found in all proton-translocating NADH:ubiquinone oxidoreductases studied thus far, it is comprised of a large number of subunits relative to the number that comprise other respiratory enzymes, and it contains flavin mononucleotide and FeS center prosthetic groups. Additionally, it possesses an L-shaped topology (14, 22) like that of its Neurospora crassa homolog (27), and it consists of distinct fragments or subcomplexes. Whereas eukaryotic complex I can be dissected into a peripheral arm and a membrane arm, the E. coli enzyme consists of three subcomplexes referred to as the peripheral, connecting, and membrane fragments (29) (Fig. ​(Fig.1A).1A). The subunit composition of these three fragments correlates approximately with the organization of the 14 structural genes (nuoA to nuoN) (49) of the nuo (for NADH:ubiquinone oxidoreductase) locus (Fig. ​(Fig.1B),1B), an organization that is conserved in several other bacteria, including Salmonella typhimurium (3), Paracoccus denitrificans (53), Rhodobacter capsulatus (12), and Thermus thermophilus (54). The 5′ half of the locus contains a promoter (nuoP), previously identified and located upstream of nuoA (8, 49), and the majority of genes that encode subunits homologous to the nucleus-encoded subunits of eukaryotic complex I and to subunits of the Alcaligenes eutrophus NAD-reducing hydrogenase (17, 29, 30, 49). In contrast, the 3′ half contains the majority of the genes that encode subunits homologous to the mitochondrion-encoded subunits of eukaryotic complex I and to subunits of the E. coli formate-hydrogen lyase complex (17, 29, 30, 49). Whereas the nuclear homologs NuoE, NuoF, and NuoG constitute the peripheral fragment (also referred to as the NADH dehydrogenase fragment [NDF]), the nuclear homologs NuoB, NuoC, NuoD, and NuoI constitute the connecting fragment. The mitochondrial homologs NuoA, NuoH, NuoJ, NuoK, NuoL, NuoM, and NuoN constitute the membrane fragment (29). E. coli complex I likely evolved by fusion of preexisting protein assemblies constituting modules for electron transfer and proton translocation (17–19, 30). Open in a separate windowFIG. 1Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).Because of its smaller size and relative simplicity, researchers recently have begun to utilize complex I of E. coli, and that of its close relative S. typhimurium, to identify and characterize the mechanism(s) by which cells regulate the synthesis and assembly of this large respiratory complex (3, 8, 46) and to investigate the diverse physiological consequences caused by defects in this enzyme (4, 6, 10, 40, 59). Such defects affect the ability of cells to perform chemotaxis (40), to grow on certain carbon sources (4, 6, 10, 40, 57), to survive stationary phase (59), to perform energy-dependent proteolysis (4), to regulate the expression of at least one gene (32), and to maintain virulence (5).To begin dissecting the processes by which E. coli cells regulate the expression of nuo and the assembly of complex I, we undertook a genetic analysis of the nuo locus. Here, we present the results of studies, performed on an isogenic collection of nuo mutants, that focus on the physiological, biochemical, and molecular consequences caused by the lack of or defects in several Nuo subunits. In particular, we present evidence that NuoG, a peripheral subunit, is essential for complex I function and that it plays a role in the regulation of nuo expression and/or the assembly of complex I.     

Complex I of Rhodobacter capsulatus and its role in reverted electron transport

The activities of NAD⁺-photoreduction and NADH/decyl-ubiquinone reductase in membrane preparations of Rhodobacter capsulatus changed to the same extent under different conditions. These results indicated that NADH:ubiquinone oxidoreductase (complex I) catalyzes the electron transport in the downhill direction (respiratory chain) and in the uphill direction (reverted electron flow). This conclusion was confirmed by the characterization of a complex-I-deficient mutant of R. capsulatus. The mutant was not able to reduce NAD⁺ in the light. Since this mutant was not able to grow photoautotrophically, we concluded that complex I is the enzyme that catalyzes the reverted electron flow to NAD⁺ to provide reduction equivalents for CO₂ fixation. Complex I is not essential for the reverted electron flow to nitrogenase since the mutant grew under nitrogen-fixing conditions. As shown by immunological means, NuoE, a subunit of complex I from R. capsulatus having an extended C-terminus, was modified depending on the nitrogen source present in the growth medium. When the organism used N₂ instead of NH₄ ⁺, a smaller NuoE polypeptide was synthesized. The complex-I-deficient mutant was not able to modify NuoE. The function of the modification is discussed. Received: 28 February 1997 / Accepted: 28 August 1997     

Na+ translocation by the NADH:ubiquinone oxidoreductase (complex I) from Klebsiella pneumoniae

Complex I is the site for electrons entering the respiratory chain and therefore of prime importance for the conservation of cell energy. It is generally accepted that the complex I-catalysed oxidation of NADH by ubiquinone is coupled specifically to proton translocation across the membrane. In variance to this view, we show here that complex I of Klebsiella pneumoniae operates as a primary Na+ pump. Membranes from Klebsiella pneumoniae catalysed Na+-stimulated electron transfer from NADH or deaminoNADH to ubiquinone-1 (0.1-0.2 micromol min-1 mg-1). Upon NADH or deaminoNADH oxidation, Na+ ions were transported into the lumen of inverted membrane vesicles. Rate and extent of Na+ transport were significantly enhanced by the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP) to values of approximately 0.2 micromol min-1 mg-1 protein. This characterizes the responsible enzyme as a primary Na+ pump. The uptake of sodium ions was severely inhibited by the complex I-specific inhibitor rotenone with deaminoNADH or NADH as substrate. N-terminal amino acid sequence analyses of the partially purified Na+-stimulated NADH:ubiquinone oxidoreductase from K. pneumoniae revealed that two polypeptides were highly similar to the NuoF and NuoG subunits from the H+-translocating NADH:ubiquinone oxidoreductases from enterobacteria.     

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO₂ conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO₂ growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.     

Intact Functional Fourteen-subunit Respiratory Membrane-bound [NiFe]-Hydrogenase Complex of the Hyperthermophilic Archaeon Pyrococcus furiosus

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO₂, and H₂, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H₂ production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na⁺/H⁺ antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H₂ with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O₂ sensitivities of the two forms were similar (half-lives of ∼15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H₂ and pump Na⁺ ions.     

The respiratory complex I (NDH I) from Klebsiella pneumoniae,a sodium pump

The electrogenic NADH:Q oxidoreductase from the enterobacterium Klebsiella pneumoniae transports Na(+) ions. The complex was purified with an increase of the specific Na(+) transport activity from 0.2 micromol min(-1) mg(-1) in native membrane vesicles to 4.7 micromol min(-1) mg(-1) in reconstituted enzyme specimens. The subunit pattern resembled that of complex I from Escherichia coli, and two prominent polypeptides were identified as the NuoF and NuoG subunits of complex I. During purification the typical cofactors of complex I were enriched to yield approximately 17 nmol mg(-1) iron, 24 nmol mg(-1) acid-labile sulfide, and 0.79 nmol mg(-1) FMN in the purified sample. The enzyme contained approximately 1.2 nmol mg(-1) Q6 and 1.5 nmol mg(-1) Q8. The reduction of ubiquinone by NADH was Na(+)-dependent, which indicates coupling of the chemical and the vectorial reaction of the pump. The Na(+) activation profile corresponded to the Hill equation with a Hill coefficient K(H)(Na(+)) = 1.96 and with a half-maximal saturation at 0.33 mm Na(+). The reconstituted complex I from Klebsiella pneumoniae catalyzed deamino-NADH oxidation, Q1 reduction, and Na(+) translocation with specific activities of 2.6 units mg(-1), 2.4 units mg(-1), and 4.7 units mg(-1), respectively, which indicate a Na(+)/electron stoichiometry of one.     

Change of Haemolymph Ferritin of Sweet Potato Hornworm, Agrius convolvuli by Heavy Metals

Agrius convolvuli haemolymph ferritin was purified by KBr density gradient ultracentrifugation and anion exchange column chromatography. The 670 kDa ferritin was composed of two subunits of 26 kDa and 31 kDa. It was also shown that the protein had an isoelectric point (pI) of pH 7.4. The N‐terminal amino acid sequences of the two subunits were NH2‐DNXYQDVSLDXSQAXNXL (26 kDa subunit) and NH2‐TQXHVNPVNIQRDXVTMHXS (31 kDa subunit). The sequential analysis showed that they had high similarity to lepidopteran ferritin subunits, S‐ and G‐type, respectively. Using electron microscope, it was observed that the protein had a core whose size was about 7 nm. In the amino acid composition of the protein, Glu (13.22%), Asp (10.43%), Pro (9.69%), Leu (9.63%), Ala (9.55%) and Gly (8.49%) were in relatively high contents while Tyr (1.21%), His (2.58%) and Arg (3.10 %) were in low. It was shown that the amount of ferritin in A. convolvuli haemolymph was increased by injection of eight different heavy metal ions, FeCl₃, HgCl₂, CuSO₄, ZnSO₄, MnCl₂, MgCl₂, CrCl₃ and CdCl₂. Among the ions, Fe³⁺, Hg²⁺, Zn⁴⁺, Mn²⁺ and Cd.²⁺ significantly induced the amount of the protein.     

1H, 13C,and 15N backbone resonance assignments of the 37 kDa voltage-gated Ca2+ channel β4 subunit core SH3-GK domains

The β subunit of the voltage-gated Ca²⁺ channel (α₁, α₂δ, and β subunits) is a member of the MAGUK family of proteins and plays an essential role in regulating Ca²⁺ channel trafficking and gating. It also serves as a central interaction partner for various Ca²⁺ channel regulatory proteins. We report here the nearly complete ¹H, ¹³C, and ¹⁵N backbone resonance assignments of the 37 kDa core SH3-GK domains of the β4 subunit. This is the first report of solution assignments for β subunits, and as such will lay the foundation for future investigations of interaction site mapping, functional dynamics, and protein complex structure determination.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Iron-sulfur cluster N7 of the NADH:ubiquinone oxidoreductase (complex I) is essential for stability but not involved in electron transfer

The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli is composed of 13 subunits called NuoA through NuoN. It catalyzes the electron transfer from NADH to ubiquinone by a chain of redox groups consisting of one FMN and seven iron-sulfur clusters. The function of the additional, nonconserved cluster N7 located on NuoG is not known. It has been speculated that it is not involved in electron transfer, due to its distance of more than 20 A from the electron transfer chain. Dithionite-reduced minus NADH-reduced EPR difference spectra of complex I and of a soluble fragment containing NuoG revealed for the first time the EPR spectrum of N7 in the complex. Individual mutation of the cysteines ligating this cluster to alanine led to a decreased amount of complex I in the membrane without affecting the electron transfer activity. Sucrose gradient centrifugation revealed that the complex from the C230A and C233A mutants decayed in detergent solution while the C237A and C265A mutant complex was stable. Cluster N7 was detectable in the latter mutants but with shifted g-values, indicating a different ligation of N7. Thus, N7 is essential for the stability of the complex but is not involved in electron transfer.     

Purification and Properties of the F1Fo ATPase of Ilyobacter tartaricus, a Sodium Ion Pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F₁F_o ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, , and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na⁺-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na⁺ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na⁺ or Li⁺ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na⁺, Li⁺, or H⁺. Proton transport was specifically inhibited by Na⁺ or Li⁺ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na⁺ as the physiological coupling ion for ATP synthesis.     

The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping

The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.