Significance of [2Fe-2S] Cluster N1a for Electron Transfer and

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Significance of the [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I Katerina Dörner, Marta Vranas, Johannes Schimpf, Isabella R. Straub, Jo Hoeser, and Thorsten Friedrich Biochemistry, Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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Significance of the [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I † † This work is supported by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School) and by the Deutsche Forschungsgemeinschaft by grants to TF (GRK 1976, GRK 2202 and FR-1140/11-1) Katerina Dörner#, Marta Vranas+,°, Johannes Schimpf, Isabella R. Straub†, Jo Hoeser and Thorsten Friedrich+,*

Institut für Biochemie, Albert-Ludwigs-Universität, Albertstraße 21, 79104 Freiburg, Germany and +Spemann Graduate School of Biology and Medicine (SGBM), AlbertLudwigs-University Freiburg

RECEIVED DATE Running Title: Role of Fe-S cluster N1a in complex I #

Present address: European XFEL GmbH, Holzkoppel 4, Schenefeld, Germany

° Present address: Department of Pharmacology & Therapeutics, McGill University, 3655 Prom. Sir William Osler, Montréal, Canada †

Present address: Department of Molecular Neurogenetics, McGill University, 3801 Rue

University, Montréal, Canada * Corresponding author:

phone : +49-(0)761-203-6060, fax : +49-(0)761-203-6096 [email protected]

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Abbreviations: Complex I, proton-pumping NADH:ubiquinone oxidoreductase; d-NADH, deamino-NADH; EPR, electron paramagnetic resonance; FMN, flavin mononucleotide; Fe-S, iron-sulfur; IDA, iminodiacetic acid; MES, 2-(N-morpholino)-ethanesulfonic acid

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ABSTRACT

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. The binuclear Fe-S cluster N1a is not part of this long chain but is located in electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes while the amount of the complex was only slightly diminished. N1a was not detectable by EPR spectroscopy and, unexpectedly, the content of the binuclear cluster N1b located on a neighboring subunit was significantly decreased. Due to the lack of N1a and the partial loss of N1b the variants did not survive detergent extraction from the mutant membranes. Only the C97AE variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. It is discussed whether N1a is involved in the physiological electron transfer reaction.

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NADH:ubiquinone oxidoreductase, respiratory complex I, is the entry point of electrons into the respiratory chains of most eukaryotes and many bacteria. It couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane.1-4 Mitochondrial complex I consists of 45 subunits with a molecular weight of about 1 MDa,5 while the bacterial complex is generally composed of 14 subunits resulting in a molecular mass of approximately 530 kDa.6 Bacterial and mitochondrial complex I contain the same cofactors, namely one non-covalently bound flavin mononucleotide (FMN), two binuclear iron-sulfur (Fe-S) clusters named N1a and N1b and six conserved tetranuclear Fe-S clusters N2, N3, N4, and 4Fe[NuoG]C, 4Fe[NuoG]H and 4Fe[NuoI]2 (Fig. 1).1-6 The assignment of the structurally defined clusters to the EPR-spectroscopic signals is under discussion.7,8 Here, we use a preliminary nomenclature to avoid confusion.8 Complex I has a L-shaped appearance consisting of a peripheral arm made up by globular subunits and a membrane arm composed of polytopic subunits. The peripheral arm catalyzes electron transfer and the membrane arm proton translocation. The structure of the complex was determined at molecular resolution by x-ray crystallography and cryo-electron microscopy.9-11 Seven out of the eight Fe-S clusters comprise a 95 Å long electron transfer chain connecting the two substrate binding-sites (Fig. 1).5,9,10,12 The eighth cluster N1a, however, is not part of the long electron transfer chain, although it is strictly conserved. N1a is located in electron tunneling distance to the flavin at the opposite site of the chain of Fe-S clusters. Hence, electrons can be transferred between FMN and N1a, but not directly between N1a and the chain of clusters.13,14 The primary electron acceptor FMN converts the hydride transfer from NADH into subsequent one-electron transfers from the FMNH2 to the Fe-S clusters adopting an intermediary radical state with a low stability constant.15-17 When the last cluster of the chain, N2, is oxidized, both electrons travel along the chain to N2. When N2 is reduced, the first electron from FMNH2 reduces either N1b or N4 forming a flavin radical, FMNH•, that 4


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transfers the second electron mainly to N1a. The production of superoxide that depends on the redox state of FMN is, thus, regulated by the redox state of the Fe-S clusters.17-19 It was also proposed that N1a might solely be important for the stability and the assembly of the complex because it is not reduced by NADH in complex I from most species.20; but see 21 Cysteine residues C92, C97, C133 and C137 of subunit NuoE coordinate N1a in Escherichia coli complex I. In this work, the cysteine residues were individually replaced by alanine and serine residues. Amounts of the variant proteins and their enzymatic activity in the membrane were determined. The stability of the variants was tested by sucrose gradient centrifugation and their purification was attempted. From our results we conclude that N1a confers stability to E. coli complex I and is unexpectedly involved in the incorporation of cluster N1b.

MATERIAL AND METHODS Materials and Strains. E. coli strains DH5α∆nuo12, BW25113∆nuo22 were generated by deletion of the nuo-operon in the DH5α and BW25113 strains, respectively. E. coli strain BW25113∆nuo∆ndh was generated by chromosomal deletion of ndh coding the alternative NADH dehydrogenase in strain BW25113∆nuo (M. Vranas, D. Kreuzer Deković and Th. Friedrich, unpub. results). Strain DH5α (Invitrogen), the plasmids pBADnuo nuoFHis23, containing the nuo-operon and coding a His-tag N-terminal on NuoF, pCA24NnuoE24 coding for the single subunit NuoE, and pVO110025 and pKD4626 were used. Ampicillin (100 µg/mL), chloramphenicol (170 µg/mL) and kanamycin (50 µg/mL) were added when necessary. All enzymes used for recombinant DNA techniques were from Fermentas (St. Leon-Roth). DNA oligonucleotides were from MWG Operon (Ebersberg, Germany). Site-directed mutagenesis. Mutations in nuoE were generated using the QuikChange method (Stratagene). The sequences of the oligonucleotides used to introduce the desired

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mutations are listed in Table S1 (Supporting Information). The plasmid pCA24NnuoE was used as template. All mutations were confirmed by DNA sequencing. λ-Red-mediated recombineering. In the first recombination step, nuoE on pBADnuo nuoFHis was replaced by the nptI-sacRB-cartridge. The cartridge was amplified with the primer pair nuoE:nptI-sacRB (Supporting Information, Table S2) from the plasmid pVO1100. DH5α∆nuo/pKD46 was co-transformed with 50 ng pBADnuo nuoFHis and 400 ng of the PCR product and recombinants were selected on LB-agar supplemented with kanamycin and chloramphenicol. The plasmids were isolated and characterized by restriction analysis. The primer pair nuoEforw and nuoErev (Supporting Information, Table S2) was used to amplify fragments of nuoE coding for the mutations C92A, C97A, C97S, C133A, C133S and C137A from pCA24NnuoE. In a second recombination step, DH5α∆nuo/pKD46 was co-transformed with 50 ng pBADnuo nuoFHis nuoE::nptI-sacRB and 400 ng of the desired PCR product. Recombinants were selected on LB-agar supplemented with 10% (w/v) sucrose and chloramphenicol. The plasmids were isolated and screened by restriction analysis. Individual mutations were confirmed by DNA sequencing. Cell growth and membrane isolation. E. coli cells from strains BW25113∆nuo and BW25113∆nuo∆ndh were individually transformed with pBADnuo nuoFHis and with the plasmids containing the desired mutation. Parental and mutant strains were grown aerobically in 400 mL cultures containing 1% (w/v) peptone, 0.5% (w/v) yeast extract, 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 0.5 mM L-cysteine, 30 µg/mL Fe-NH4-citrate, 50 mg/mL riboflavin, 0.5% (w/v) D-mannitol, 0.2% (w/v) L(+)arabinose and 0.05% (w/v) glucose. Cells were harvested by centrifugation at 10’810g at 4°C and stored at -80°C until use. Cytoplasmic membranes were prepared at 4°C. 5 - 10 g cells were suspended in the five fold volume 50 mM MES/NaOH, 50 mM NaCl, 0.1 mM phenylmethane sulfonyl fluoride (PMSF), pH 6.0, with 10 µg/mL DNase I and disrupted by a single pass through a French pressure cell (SLM Amico) at 110 MPa. Cell debris was 6


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removed by centrifugation for 20 min at 14’000g and 4°C. Cytoplasmic membranes were obtained by centrifugation of the supernatant for 1 h at 250’000g. The membranes were suspended in 50 mM MES/NaOH, 50 mM NaCl, 0.1 mM PMSF, pH 6.0 and stored on ice. Sucrose gradient centrifugation. Membrane proteins were separated by sucrose gradient centrifugation as described.25 All steps were carried out at 4°C. Generally, the membrane fraction was obtained as described above and was mixed with n-dodecyl-ß-D-maltopyranoside (DDM, BioFroxx) to a final concentration of 3% (w/v) and centrifuged for 20 min at 250’000g and 4°C. The supernatant containing 10 to 20 mg protein was applied onto a 12 mL sucrose gradient and proteins were separated by centrifugation for 18 h at 180’000g and 4°C. The gradient was fractionated in 600 µL portions and the fractions were tested for NADH/ferricyanide oxidoreductase activity and used for western blot analysis. Purification of complex I. Complex I and the C97AE variant were isolated from E. coli strain BW25113∆nuo∆ndh transformed with the corresponding plasmid as described.23 Briefly, membrane proteins were extracted using DDM and separated by anion exchange chromatography

on

Fractogel

EMD

TMAE

Hicap

(Merck).

Fractions

with

NADH/ferricyanide oxidoreductase activity were pooled and further purified by affinity chromatography on Ni-IDA material (Invitrogen). Bound proteins were eluted in an imidazole gradient. Complex I eluted at 260 mM imidazole. In addition to what has been described,23 fractions from the affinity chromatography containing complex I were further purified on a 15 mL Resource Q (GE Healthcare) column equilibrated in 50 mM MES/NaOH, 50 mM NaCl, 0.1% DDM, pH 6.0 at a flow rate of 1.5 mL/min. Bound proteins were eluted in a 40 mL linear gradient from 150-350 mM NaCl in 50 mM MES/NaOH, 0.1% DDM, pH 6.0. Peak fractions with NADH/ferricyanide oxidoreductase activity were combined and concentrated by ultrafiltration (Amicon Ultra-15, Millipore, 100 kDa MWCO) and stored in aliquots at 80°C.

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Analytical assays. The (d-)NADH oxidase activity of complex I in cytoplasmic membranes was measured with a Clark-type oxygen electrode as described.27 The (d)-NADH/ferricyanide oxidoreductase activity was determined as described.28 EPR measurements were conducted with an Elexsys E500 X-band spectrometer equipped with an Oxford Instruments helium flow cryostat. The magnetic field was calibrated using a strong pitch standard. The individual experimental conditions are provided in the figure legends. Protein concentration was determined by the biuret method using BSA as standard. SDS-PAGE was performed according to Schägger and von Jagow.29 Subsequently, proteins were electroblotted onto 0.45 µm pore size PVDF membrane (Schleicher and Schüll) according to Towbin et al.30 Rabbit polyclonal antibodies raised against isolated complex I subunits were kindly provided by Dr. Takao Yagi, La Yolla, USA. The bands were detected with an IRDye 680CW conjugated Goat anti-rabbit IgG as secondary antibody (Li-cor).

RESULTS Growth of the nuoE mutants and catalytic activity of the membranes from strain BW25113∆nuo. Mutations within the N1a binding motif were introduced into the plasmid pBADnuo nuoFHis by λ-Red-mediated recombineering and confirmed by DNA sequencing. E. coli strain BW25113∆nuo was transformed with the recombinant plasmids. Growth of the mutant strains in phosphate-buffered LB-medium was indistinguishable to that of the parental strain BW25113∆nuo/ pBADnuo nuoFHis due to the presence of the alternative, non-energyconverting NADH dehydrogenase (ndh) in the respiratory chain of E. coli strain BW25113∆nuo. The alternative NADH dehydrogenase has a thousand fold lower affinity to the artificial substrate d-NADH than complex I,27 therefore, d-NADH was used to determine complex I activity in cytoplasmic membranes. The d-NADH/ferricyanide oxidoreductase and the d-NADH oxidase activities of membranes from strain BW25113∆nuo and the nuoE mutants are listed in Table 1. 8


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All mutations resulted in a significant decrease of the artificial d-NADH/ferricyanide oxidoreductase activity (Table 1). The variants exhibited 19 to 44% of the activity of the membranes from the parental strain. Thus, the mutations might affect the assembly and stability of the complex or they might decrease its NADH dehydrogenase activity. Noteworthy, the Fe-S clusters are involved in the NADH/ferricyanide oxidoreductase activity, 15,17

therefore, the mutations might affect the rate of the reaction. Concomitant with the d-

NADH/ferricyanide oxidoreductase activity, the physiological d-NADH oxidase activity of the mutant membranes was diminished to 13 - 37% of that of the membranes from the parental strain (Table 1). As the d-NADH oxidase activity of the mutant membranes was fully sensitive to piericidin A, a specific complex I inhibitor, it is evident that the oxidase activity was mediated by complex I. Whereas d-NADH oxidase activity was detectable in membranes from all mutant strains containing a complex I variant, the membranes from the strain transformed with the empty plasmid did not show any d-NADH oxidase activity (Table 1). Thus, the NuoE mutants exhibited a low but distinct complex I activity. The ratio between the oxidase and the ferricyanide oxidoreductase activity, commonly a measure for the share of complex I with an active flavin site in the membranes, did not show significant changes between the parental strain and the variants (Table 1). The values have to be taken with care as mutations around N1a might also alter the NADH/ferricyanide activity. To determine the amount of the variants in the mutant membranes western blot analysis was used.

Amount of complex I in the mutant membranes. Cytoplasmic membranes from the parental strain and from the mutants were washed with buffer and loaded on an SDS-PAGE. The separated proteins were transferred onto a PVDF membrane. Subunit NuoF was detected by western blot analysis using a polyclonal antibody. Figure 2 shows the results obtained with the C137AE and C97AE mutants exhibiting the highest and lowest d-NADH oxidase activity, respectively (Table 1). Quantification of the secondary antibody’s fluorescence signal 9


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revealed that the membranes of the various strains contained a similar amount of NuoF. The signal obtained for the parental strain was used as reference and set to 100%. The signal intensity of the membranes from the C137AE mutant strain resulted in 75%, and from the C97AE mutant strain in 80% of the parental strain. Similar results were obtained with the other mutants. NuoF is a subunit of the peripheral arm of complex I containing FMN and the NADH binding-site. Its presence in washed cytoplasmic membranes from the parental strain and the mutants in almost equal shares indicates the full assembly of the complex in the mutant strains. Loss of the adjacent NuoE would lead to a disturbed assembly of complex I resulting in the lack of membrane-attached NuoF.31 As the d-NADH oxidase activity of the mutants is decreased by an average of 75% (Tab. 1) whereas the protein content is diminished by no more than 25% (Fig. 2), it is reasonable to assume that the variants show a strongly reduced NADH dehydrogenase activity. The mutations might lead to the presence of a nonfunctional complex probably resulting in a disturbed growth of mutant strains that also lack the alternative NADH dehydrogenase.

Growth of the nuoE mutants and catalytic activity of the membranes from strain BW25113∆nuo∆ndh. E. coli strain BW25113∆nuo∆ndh lacking the nuo-genes coding complex I and the gene ndh coding the alternative NADH dehydrogenase was individually transformed with the plasmids coding complex I and the NuoE variants. The mutant strains grew significantly slower in phosphate-buffered LB-medium than the parental strain (Fig. 3). The C133AE and C133SE mutant strains grew very slowly, while the C92AE, C97AE, C137AE and C97SE mutant strains showed intermediate growth rates (Fig. 3). Thus, the NuoE mutations significantly diminished the growth rates of E. coli cells lacking the alternative NADH dehydrogenase indicating the presence of a non-functional complex in the mutant membranes.

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The ∆nuo∆ndh double deletion strains were also used to determine complex I activities in the membrane as described above. Due to the lack of the alternative NADH dehydrogenase, the cost-effective NADH was used as substrate for this assay (Table 2). The results obtained with the double deletion strains were in accordance with the data obtained from the nuodeletion strains (Table 1) with a similar impact of the individual mutations on the NADH/ferricyanide oxidoreductase and NADH oxidase activities. The NADH/ferricyanide oxidoreductase activity is reduced to 7 - 25% to that of the membranes from the parental strain. The NADH oxidase activity of the NuoE variants in the ∆nuo∆ndh double deletion strain is similarly diminished to 8 - 24% (Table 2). The NADH oxidase activity of membranes from the NuoE variants was clearly detectable and inhibited by piericidin A. Membranes from the strain transformed with the empty plasmid did not show NADH oxidase activity (Table 2).

Presence of cluster N1a in the mutant membranes. The diminished NADH oxidase activity observed with the mutant membranes might be due to a loss of cluster N1a. Therefore, EPR spectroscopy was used to examine the presence of the binuclear Fe-S clusters of complex I in the mutant membranes. Due to the presence of several membrane-bound proteins containing binuclear Fe-S clusters, it is not possible to detect N1a in a spectrum of membranes that have been chemically reduced e.g. by an addition of dithionite. Due to the spectral overlap with other clusters it is also not possible to detect all tetranuclear Fe-S clusters of complex I in the membrane. The experiment is further hampered by the presence of strong signals from membrane-bound manganese ions from the growth medium. To circumvent these problems difference spectroscopy was applied. NADH was added to one aliquot of the membrane suspension to specifically reduce the cofactors of complex I while another aliquot was diluted with the same volume of buffer. Spectra were recorded at 40 K and 5 mW microwave power to detect the binuclear Fe-S clusters. The spectrum of the membranes in the air-oxidized state was subtracted from that of the membranes reduced by NADH. The (NADH-reduced minus 11


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air-oxidized) difference spectrum of the membranes from the parental strain clearly showed all signals of clusters N1a (gz = 1.999; gy = 1.95; gx = 1.925) and N1b (gz = 2.028; gy,x = 1.94; Fig. 4A) demonstrating that the method is well suited to detect the binuclear Fe-S clusters of complex I. Membranes from the C97AE mutant showed a difference spectrum with signals that were attributed to N1a and N1b but with shifted g-values. The signals of N1b were slightly shifted (gz = 2.029; gy,x = 1.95), but the signals of N1a were prominently shifted to gz = 2.011; gy = 1.94 and gx = 1.924 (Fig. 4B). The relative amount of N1a and N1b in the membranes from the parental strain and the C97AE mutant strain was estimated to 1:0.75 each from a linear combination of the simulated spectra (Fig. 4). The amount of the clusters in the mutant membranes was approximately 20% of that of the membranes from the parental strain. Thus, the C97AE mutant lacked not only N1a but also N1b located on NuoG in an approximately equal share. No clear signal from N1a was detectable in the membranes of all other mutants. The amount of N1b in the mutant membranes is less than 20% of that of membranes from the parental strain. The difference spectra contained a radical signal hampering the detection in the gz region of the N1a signal. However, due to the radical signal being symmetrical and no visible signal in the gx region of N1a from 1.92 to 1.93 it is rather unlikely that N1a is present in these variants. Nevertheless, a very minor amount of N1a in the mutant membrane would not be detectable with the method applied. The signals from N1b were detected in very minor amounts at positions shifted to gz = 2.022 and gy,x = 1.94 (Fig. 4C). While cluster N1a is present in the C97AE mutant but it is not detectable in the other mutants, the NADH oxidase activity does not significantly differ between the strains. This might indicate that N1a is not essential for the physiological NADH:ubiquinone oxidoreductase activity of complex I, or at least not when the quinone pool is mainly in the oxidized state.17

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Stability of the complex I variants. The stability of the variants was probed by detergent extraction of membrane proteins followed by ultracentrifugation in a sucrose gradient. Fractions of the gradient loaded with extracts from cytoplasmic membranes of the parental strain BW25113∆nuo and of the NuoE mutant strains were assayed for NADH/ferricyanide oxidoreductase activity (Fig. 5). The activity in fractions 5 and 6 is assigned to the alternative NADH dehydrogenase25,28 present in these strains. The enzyme is produced in similar amounts in all strains, as roughly the same activity was present in the corresponding fractions of the extracts from all mutant membranes. The activity maximum around fraction 14 observed with the extract from strain BW25113∆nuo/pBADnuo nuoFHis is attributed to complex I.25,28 It was not possible to detect any NADH/ferricyanide oxidoreductase activity around fraction 14 in the membrane extract from most mutant strains (Fig. 5). However, a small but reproducible peak was obtained in the extract from the membranes of strain pBADnuo nuoFHis nuoE C97A (Fig. 5). To determine the amount of complex I in the fractions 13 to 15 of the gradient, western blot analysis was applied as described above. Clear signals from subunits NuoF and NuoE were detected in the fractions of the gradient obtained with the parental strain, while only signals with diminished intensities were detected in the gradients loaded with extracts from the mutant membranes (Fig. 6). Normalizing the signal intensity with the signal obtained from the extract of the parental strain, 14% of NuoE and 16% of NuoF were present in the extract from the C97AE mutant and 14% of NuoE and 15% of NuoF in that of the C137AE mutant, respectively. Thus, the mutant strains do not contain a stable complex I. Most likely, the so-called NADH dehydrogenase fragment of the complex consisting of NuoE, F, and G was lost for the most part during the extraction and centrifugation.6 The residual amount of the variant complexes in the fractions of the gradient was too low to be detected by NADH/ferricyanide oxidoreductase activity.

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Preparation of the C97AE variant. It was obvious that the C97AE mutant contained a complex I variant that differed from that in the other strains regarding the presence of N1a (Fig. 4B). In addition, a small peak of complex I activity was detected in the sucrose gradient at a position typical for a fully assembled complex (Fig. 5). For this reason, purification of the variant was attempted from the mutant membranes with the established protocol.23 Starting from 44 g cells (wet weight) of strain BW25113∆nuo/ pBADnuo nuoFHis C97AE 2.4 mg protein were obtained. Routinely, 7 mg complex I are obtained from the preparation of the complex from 30 g cells (wet weight) of the parental strain.32 While the NADH:decylubiquinone oxidoreductase activity of both preparations was 3.3 U mg-1 in the presence of 50 µM NADH and 100 µM decyl-ubiquinone, the NADH/ferricyanide oxidoreductase activity of the variant was slightly lower compared to complex I. Titrating of the NADH/ferricyanide oxidoreductase activity with various amounts NADH resulted in a maximum turnover of 550 ± 30 NADH per second with complex I while that of the C97AE variant was 450 ± 20 NADH per second. Thus, the rate of the very fast NADH oxidation was slightly reduced in the variant, without becoming rate limiting for the much slower NADH:decyl-ubiquinone oxidoreductase activity. The preparation was reduced by NADH and characterized by EPR spectroscopy (Fig. 7). The spectrum of a complex I preparation exhibits the well-known g-values of N1a (gz = 1.999; gy = 1.95; gx = 1.925) and N1b (gz = 2.028; gy,x = 1.94). In the spectrum of the C97AE variant the signals of N1b remained unchanged, while the signals of N1a were clearly shifted to gz = 1.997; gy = 1.97 and gx = 1.923 (Fig. 7). The signals are significantly broadened, especially the central gy absorbance. Similar findings were obtained with variants of the overproduced single subunit from Paracoccus denitrificans.33 The relative spin concentration was N1a:N1b = 1.0:0.9 in complex I and in the variant. Thus, the C97AE variant contains N1a with clear changes in its local environment, while the environment of cluster N1b remains unchanged. 14


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DISCUSSION The relevance of Fe-S cluster N1a to the mechanism of complex I is not understood. The cluster is not part of the chain of Fe-S clusters connecting the NADH oxidation site with the quinone reduction site.34 However, it is fully conserved in the complex from all species investigated so far. It was proposed that it takes up one electron from the FMNH• intermediate to reduce the likelihood of producing reactive oxygen species (ROS).35 Indeed, it was experimentally demonstrated that in E. coli the FMNH•/FMN redox couple reduces N1a when the distal cluster of the chain is in the reduced state.17 However, it is now generally accepted that the fully reduced FMNH2 is the source of ROS, at least in mitochondria.36 Furthermore, the redox properties of N1a remain enigmatic. N1a has a redox potential in the range from 250 to -295 mV in complex I from E. coli and Aquifex aeolicus.37,38 Accordingly, it is reduced by NADH in isolated E. coli complex I and in a soluble flavoprotein subcomplex from E. coli and A. aeolicus.39,40 N1a is also reduced by NADH in a flavoprotein subcomplex of bovine heart complex I,41 but not in the entire complex.42 It is also not reduced by NADH in the entire complex from Bos taurus, Yarrowia lipolytica, Pichia pastoris, P. denitrificans and Thermus thermophilus.43-46 It is discussed that this is most likely due to the low redox potential of N1a in the complex from these organisms in the range from -370 to -420 mV. However, it is puzzling that N1a is not reduced in the B. taurus complex I by an Eu(II) reagent with a very low potential of about -1 V41 while it is in electron tunneling distance to flavin.34 This might indicate that N1a is not accessible from the solution and from the flavin in the holo-complex.40 Manipulating the redox potential of N1a by site-directed mutagenesis to less than -430 mV led to an E. coli variant in which N1a was no longer fully reducible with NADH.20 However, the NADH/ferricyanide oxidoreductase and the NADH oxidase activities of the mutant were only slightly diminished by approximately 20%. For that reason, it is discussed that N1a has solely a structural role in complex I or is needed for its assembly.20 15


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Here, we showed that individual substitution of residues C92E, C97E, C133E and C137E from the N1a binding motif of E. coli complex I by an alanine or a serine residue led to a strongly diminished (d-)NADH/ferricyanide oxidoreductase and (d-)NADH oxidase activity in strains either containing or lacking the alternative NADH dehydrogenase (Tables 1 and 2). However, the NuoE variants exhibited some activity that was fully sensitive to piericidin A, while the mutant completely lacking complex I and the alternative NADH dehydrogenase showed no NADH oxidase activity (Table 2). The NADH oxidase activity in the mutants was reduced by 75%, but the amount of NuoF in the membranes was reduced by only 20% compared to the parental strain (Fig. 2). Cluster N1a was not traceable in most mutant membranes. Thus, it is unlikely but cannot be excluded that the mutants contain a residual amount of N1a. In contrast, a variant containing N1a was detected in membranes of the C97AE mutant (Fig. 4 and see below). Unexpectedly, the amount of cluster N1b in the mutant membranes was diminished by approximately 80% compared to the parental strain. From this data two main conclusions on the assembly of the complex and its electron transfer activity might be drawn. First, it is obvious that E. coli complex I is assembled in the absence of N1a. If the lack of N1a affected the assembly of NuoE into the holo-complex, NuoE and F would not be attached to the cytoplasmic membrane.31 NuoE, F and G are joint with the residual subunits of the complex in the last step of the assembly process.6 Furthermore, the inability to insert N1a into the complex also hampered the incorporation of N1b located on a separate subunit. Thus, the incorporation of N1a into NuoE might lead to structural changes facilitating the incorporation of N1b on NuoG. The NuoE variants are assembled in the membrane although they are lacking N1a and N1b. N1a seems to be indeed essential for the stability of the complex as proposed.20 While the amount of NuoF in the mutant membranes is about 80% of that of the membranes from the parental strain (Fig. 2) only 15% of NuoE and F are found in the relevant fractions of a sucrose gradient loaded with a detergent extract from the mutant membranes (Fig. 6). It is 16


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Biochemistry

reasonable to assume that binding of N1a confers a rigid structure to NuoE enabling its interaction with NuoF and G. The loss of these interactions might in turn lead to the loss of NuoE, F and G from the residual subunits and, accordingly, to the loss of NADH/ferricyanide oxidoreductase activity. Most likely, the residual subunits of the complex are still assembled in the mutant membranes but their presence cannot be addressed by the methods applied. The mutation in the N1a binding motif might lead to a disturbed assembly of NuoE, F and G as reported for mutations around the non-conserved Fe-S cluster N7 on NuoG.12 N1a was not detectable in the mutant’s membranes with the exception of the C97AE mutant (see below). If N1a is involved in the NADH oxidase activity, this activity would be strongly abolished in all mutants but not in C97AE. As this was not observed, N1a is probably not necessary for the NADH:(ubi)quinone oxidoreductase activity of complex I under steadystate conditions, i.e. when the quinone pool is sufficiently oxidized to keep the redox centers of the complex in an oxidized state. It has to be taken into account that N1a is only involved in the intramolecular electron transfer in E. coli complex I when the most distal cluster N2 is reduced.17 In contrast, the maximum turnover of the NADH/ferricyanide oxidoreductase activity is slightly but significantly diminished in the C97AE variant indicating an involvement of the cluster in this reaction. The low NADH oxidase activity of the variants might be due to the loss of N1a, to structural changes in the NADH binding site and to the diminished content of N1b. Calculations of the electron transfer rates in complex I demonstrated that the loss of N1b would lead to a ten times slower reaction from NADH to quinone but not block electron transfer.45 Mutational studies on the N1a binding motif were also reported for the heterologously overproduced single subunit Nqo2 (the NuoE homologue of P. denitrificans complex I).33 Replacing the cysteine residues individually by either alanine or serine residues led to a slight loss of the Fe-S cluster in the protein and to clear changes of its EPR-spectroscopic properties.33 Most likely, the structure of Nqo2 changed in response to the mutation and the 17


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single subunit Nqo2 variants could be isolated in a stable form. Introducing the corresponding mutations in NuoE might also cause a change in its overall structure interfering with protein/protein interactions within the entire E. coli complex I. Furthermore, the C-terminus of the P. denitrificans Nqo2 contains a C-terminal extension of 69 amino acids that possibly supports the stability of Nqo2. The g-values of N1a in the C97AE variant were strongly shifted (Fig. 7B). The structure of the T. thermophilus complex I hints to residues S88E, H98E, Y102E and C132E as possible candidates for binding N1a in the variant. A slight change in the protein structure could enable these residues to substitute C97E. It is likely that only H98E is capable of ligating N1a in the C97AE variant due to steric hindrance from the newly introduced serine residue.

ACKNOWLEDGMENT We thank Prof. Dr. Takao Yagi, The Scripps Institute, La Jolla, USA, for the kind gift of the NuoE and NuoF antibodies. We thank Prof. Peter Stäheli, Department of Virology, Albert-Ludwigs-Universität Freiburg, for the kind gift of the secondary antibody for western blot analysis.

Supporting Information Available: Oligonucleotides used for site-directed mutagenesis (Tab. S1), Oligonucleotides used for λRed-mediated recombination (Tab. S2). This material is available free of charge via the internet at http://pubs.acs.org.

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dynamic shifting of generation sites between semiflavin and semiquinone radicals. Biochim. Biophys. Acta 1797, 1901-1909. 20. Birrell, J. A., Morina, K., Bridges, H. R., Friedrich, T., and Hirst, J. (2013) Investigating the function of [2Fe-2S] cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential, Biochem. J. 456, 139-146. 21. Ohnishi, T. (1975) Thermodynamic and EPR characterization of iron-sulfur-centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart, Biochim. Biophys. Acta 387, 475-490. 22. Morina, K., Schulte, M., Hubrich, F., Dörner, K., Steimle, S., Stolpe, S., and Friedrich, T. (2011) Engineering the respiratory complex I to an energy-converting NADPH:ubiquinone oxidoreductase, J. Biol. Chem. 286, 34627-34634. 23. Pohl, T., Uhlmann, M., Kaufenstein, M., and Friedrich, T. (2007) Lambda Redmediated mutagenesis and efficient large scale affinity purification of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I). Biochemistry 46, 10694-10702. 24. Kitagawa, M., Ara, T., Arifuzzaman, M., Ioka-Nakamichi, T., Inamoto, E., Toyonaga, H., and Mori, H. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research, DNA Res. 12, 291-299. 25. Spehr, V., Schlitt, A., Scheide, D., Guenebaut, V., and Friedrich, T. (1999) Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I), Biochemistry 38, 16261-16267. 26. Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. U.S.A. 97, 6640-6645. 27. Friedrich, T., van Heek, P., Leif, H., Ohnishi, T., Forche, E., Kunze, B., Jansen, R., Trowitzsch-Kienast, W., Hofle, G., Reichenbach, H., and Weiss, H. (1994) Two 21


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binding sites of inhibitors in NADH:ubiquinone oxidoreductase (complex I), Eur. J. Biochem. 219, 691-698. 28. Flemming, D., Hellwig, P., and Friedrich, T. (2003) Involvement of tyrosines 114 and 139 of subunit NuoB in the proton pathway around cluster N2 in Escherichia coli NADH:ubiquinone oxidoreductase, J. Biol. Chem. 278, 3055-3062. 29. Schägger, H., and von Jagow, G. (1987) Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa, Anal. Biochem. 166, 368-379. 30. Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A. 1320, 217-234. 31. Erhardt, H., Steimle, S., Muders, V., Pohl, T., Walter, J., and Friedrich, T. (2012) Disruption of individual nuo-genes leads to the formation of partially assembled NADH:ubiquinone oxidoreductase (complex I) in Escherichia coli, Biochim. Biophys. Acta 1817, 863-871. 32. Steimle, S., Bajzath, C., Dörner, K., Schulte, M., Bothe, V., and Friedrich, T. (2011) Role of Subunit NuoL for Proton Translocation by Respiratory Complex I, Biochemistry 50, 3386-3393. 33. Yano, T., Sled, V. D., Ohnishi, T., and Yagi, T. (1994) Identification of amino acid residues associated with the [2Fe-2S] cluster of the 25 kDa (NQO2) subunit of the proton-translocating NADH-quinone oxidoreductase of Paracoccus denitrificans, FEBS Lett. 354, 160-164. 34. Berrisford, J. M., and Sazanov, L. A. (2009) Structural basis for the mechanism of respiratory complex I, J. Biol. Chem. 284, 29773-29783.

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35. Sazanov, L. A. (2007) Respiratory complex I: mechanistic and structural insights provided by the crystal structure of the hydrophilic domain, Biochemistry 46, 22752288. 36. Pryde, K. R., and Hirst, J. (2011) Superoxide is produced by the reduced flavin in mitochondrial complex I: a single, unified mechanism that applies during both forward and reverse electron transfer, J. Biol. Chem. 286, 18056-18065. 37. Leif, H., Sled, V. D., Ohnishi, T., Weiss, H., and Friedrich, T. (1995) Isolation and characterization of the proton-translocating NADH:ubiquinone oxidoreductase from Escherichia coli, Eur. J. Biochem. 230, 538-548. 38. Kohlstädt, M., Dörner, K., Labatzke, R., Koc, C., Hielscher, R., Schiltz, E., Einsle, O., Hellwig, P., and Friedrich, T. (2008) Heterologous production, isolation, characterization and crystallization of a soluble fragment of the NADH:Ubiquinone Oxidoreductase (Complex I) from Aquifex aeolicus, Biochemistry 47, 13036-13045. 39. Ragan, C. I., Galante, Y. M., Hatefi, Y., and Ohnishi, T. (1982) Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins, Biochemistry 21, 590-594. 40. Reda, T., Barker, C. D., and Hirst, J. (2008) Reduction of the iron-sulfur clusters in mitochondrial NADH:ubiquinone oxidoreductase (complex I) by EuII-DTPA, a very low potential reductant. Biochemistry 47, 8885-8893. 41. Maly, T., Grgic, L., Zwicker, K., Zickermann, V., Brandt, U., and Prisner, T. (2006) Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy, J. Biol. Inorg. Chem. 11, 343-350. 42. Bridges, H. R., Grgic, L., Harbour, M. E., and Hirst, J. (2009) The respiratory complexes I from the mitochondria of two Pichia species, Biochem. J. 422, 151-159. 43. Meinhardt, S. W., Kula, T., Yagi, T., Lillich, T., and Ohnishi, T. (1987) EPR characterization of the iron-sulfur clusters in the NADH: ubiquinone oxidoreductase 23


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segment of the respiratory chain in Paracoccus denitrificans, J. Biol. Chem. 262, 9147-9153. 44. Hinchliffe, P., Carroll, J., and Sazanov, L. A. (2006) Identification of a novel subunit of respiratory complex I from Thermus thermophilus, Biochemistry 45, 4413-4420. 45. Moser, C. C., Farid, T. A., Chobot, S. E., and Dutton, L. P. (2006) Electron tunneling chains of mitochondria, Biochim. Biophys. Acta 1757, 1096-1109.

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Table 1: d-NADH mediated activities in membranes of complex I NuoE variants in E. coli strain BW25113∆nuo. Inhibition of the NADH oxidase activity by 10 µM piericidin A (PicA) is indicated. BW25113∆nuo/

d-NADH/ferricyanide oxidoreductase activity

d-NADH oxidase activity

(µmol·min-1·mg-1)

Inhibition by PicA

Ratio

(%)

pBAD

0.10 ± 0.03

0

-

0

pBADnuo nuoFHis

1.60 ± 0.08

0.30 ± 0.02

95

0.19

pBADnuo nuoFHis nuoE C92A

0.49 ± 0.02

0.09 ± 0.01

92

0.18


0.61 ± 0.03

0.11 ± 0.01

94

0.18


0.41 ± 0.02

0.08 ± 0.01

88

0.20


0.28 ± 0.02

0.04 ± 0.01

88

0.14

pBADnuo nuoFHis nuoE C97S

0.50 ± 0.02

0.10 ± 0.01

90

0.20


0.69 ± 0.03

0.11 ± 0.01

88

0.16

25


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Table 2: NADH mediated activities in membranes of complex I NuoE variants in E. coli strain BW25113∆nuo∆ndh. The activities are specific for complex I due to the lack of the alternative NADH dehydrogenase. Inhibition of the NADH oxidase activity by 10 µM piericidin A (PicA) is indicated. BW25113∆nuo∆ndh/

NADH/ferricyanide oxidoreductase activity

NADH oxidase activity

(µmol·min-1·mg-1)

Inhibition by PicA

Ratio

(%)

pBAD

0.10 ± 0.04

0

-

0

pBADnuo nuoFHis

1.96 ± 0.08

0.25 ± 0.02

96

0.13


0.41 ± 0.06

0.06 ± 0.01

92

0.15


0.27 ± 0.09

0.05 ± 0.01

94

0.18


0.30 ± 0.05

0.04 ± 0.01

90

0.13


0.14 ± 0.08

0.02 ± 0.01

88

0.14


0.24 ± 0.08

0.04 ± 0.01

89

0.16


0.48 ± 0.06

0.06 ± 0.01

88

0.13

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LEGEND TO THE FIGURES

Figure 1. Scheme of the electron pathway in complex I. The denomination of the individual Fe-S clusters involved in electron transfer and the distances between them in Å are given.

Figure 2. Western blot of membranes of E. coli strains BW25113∆nuo/pBADnuo nuoFHis (WT), BW25113∆nuo/pBADnuo nuoFHis nuoE C137A (C137A) and BW25113∆nuo/ pBADnuo nuoFHis nuoE C97A (C97A). Each lane was loaded with 200 µg of membrane proteins. Western blotting was performed with an antibody raised against NuoF.

Figure 3. Cell growth of E. coli strains BW25113∆nuo∆ndh/pBADnuo nuoFHis (■), BW25113∆nuo∆ndh/pBADnuo nuoFHis nuoE C97A (○) and BW25113∆nuo∆ndh/pBADnuo nuoFHis nuoE C133A (∆) in phosphate-buffered LB-medium. Strain BW25113∆nuo∆ndh/pBADnuo nuoFHis nuoE C133S showed a growth curve similar to that of the C133AE mutant and the other mutants to that of the C97AE mutant.

Figure 4. EPR (NADH-reduced minus air-oxidized) difference spectra of membranes from the parental strain (A), strain BW25113∆nuo/pBADnuo nuoFHis nuoE C97A (B), and strain BW25113∆nuo/pBADnuo nuoFHis nuoE C137A (C) at 40 K and 5 mW microwave power. The difference spectra of the other mutant membranes were very similar to the one shown in (C). The absorptions of the individual Fe-S clusters are indicated. Cytoplasmic membranes (30 mg/mL) were reduced by 0.5 M NADH. Other EPR conditions were: microwave frequency, 9.36 GHz; modulation amplitude, 6 mT; time constant, 0.164 s; scan rate 17.9 mT/min. The gray lines correspond to a linear combination of the simulated spectra of clusters N1a and N1b in wild type: N1a (gx,y,z = 1.925; 1.95; 1.999 and Lx,y,z = 0.55; 1.0; 0.73 mT) and N1b (gx,y,z = 1.94; 1.94; 2.028 and Lx,y,z = 1.15; 1.15; 0.6 mT) in a 1:0.75 ratio and in the 27


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C97AE mutant: N1a (gx,y,z = 1.924; 1.94; 2.011 and Lx,y,z = 0.65; 1.5; 0.45 mT) and N1b (gx,y,z = 1.95; 1.95; 2.029 and Lx,y,z = 0.95; 0.95; 0.4 mT) in a 1:0.75 ratio.

Figure 5. Sucrose gradient of membrane protein extract from strains BW25113∆nuo/pBADnuo nuoFHis (♦), BW25113∆nuo/pBADnuo nuoFHis nuoE C97A (▲), and BW25113∆nuo/pBADnuo nuoFHis nuoE C133S (■). The NADH/ferricyanide oxidoreductase activity was normalized to 10 mg protein applied on each gradient. The activity profile of the extract from the other mutants showed a similar NADH/ferricyanide activity profile as that from the C133SE mutant.

Figure 6. Western blot of fractions 13 to 15 of sucrose gradients obtained from membrane extracts from E. coli strains BW25113∆nuo/pBADnuo nuoFHis (WT), BW25113∆nuo/ pBADnuo nuoFHis nuoE C137A (C137A) and BW25113∆nuo/pBADnuo nuoFHis nuoE C97A (C97A). Each lane was loaded with 200 µl of the corresponding fractions. Western blotting was performed with antibodies raised against NuoE (19 kDa) and NuoF (51 kDa), respectively.

Figure 7. EPR spectrum of the preparations of complex I (A) and the C97AE variant (B) each reduced with a 1’000-fold molar excess NADH recorded at 40 K and 5 mW microwave power. The absorptions of the individual Fe-S clusters are indicated. Other EPR conditions were: microwave frequency, 9.36 GHz; modulation amplitude, 6 mT; time constant, 0.164 s; scan rate 17.9 mT/min.

28


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Figure 1 254x190mm (96 x 96 DPI)


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Figure 3 272x208mm (300 x 300 DPI)


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Biochemistry



Biochemistry

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Biochemistry



Biochemistry

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For table of contents only 88x35mm (300 x 300 DPI)


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Significance of [2Fe-2S] Cluster N1a for Electron Transfer and

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