RANBP2 mutation causing autosomal dominant acute necrotizing encephalopathy attenuates its interaction with COX11.

PURPOSE: Autosomal dominant acute necrotizing encephalopathy (ADANE) is caused by missense mutations in the gene encoding Ran-binding protein 2 (RANBP2), a nuclear pore protein regulating mitochondrial localization and function. Previous studies have found that RANBP2 binds to COX11 and suppresses its inhibitory activity over hexokinase1. To further elucidate mitochondrial dysfunction in ADANE, we analyzed the interaction between mutated RANBP2 and COX11. METHODS: We extracted cDNA from a patient and constructed pGEX wild-type or mutant-type vectors including RANBP2 c.1754C>T, the commonest variant in ADANE. We transformed E. coli competent cells with the vectors and had them express GST-RANBP2 recombinant protein, and conducted a pull-down assay of RANBP2 and COX11. RESULTS: The amount of COX11 bound to mutated RANBP2 was significantly smaller than that bound to the wild-type RANBP2. CONCLUSION: Mutated RANBP2 had an attenuated binding ability to COX11. Whether this change indeed decreases ATP production remains to be further explored.

The carboxy-terminal insert in the Q-loop is needed for functionality of Escherichia coli cytochrome bd-I.

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes.

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer structural and functional advantages to mitochondria. SCs have been identified in many species, from yeast to mammal, and an increasing number of studies report disruption of their organization in genetic and acquired human diseases. As a result, an increasing number of laboratories are interested in analyzing SCs, which can be methodologically challenging. This article presents an optimized protocol that combines the advantages of Blue- and Clear-Native PAGE methods to resolve and analyze SCs in a time-effective manner. With this hybrid CN/BN-PAGE method, mitochondrial SCs extracted with optimal amounts of the mild detergent digitonin are exposed briefly to the anionic dye Coomassie Blue (CB) at the beginning of the electrophoresis, without exposure to other detergents. This short exposure to CB allows to separate and resolve SCs as effectively as with traditional BN-PAGE methods, while avoiding the negative impact of high CB levels on in-gel activity assays, and labile protein-protein interactions within SCs. With this protocol it is thus possible to combine precise and rapid in gel activity measurements with analytical techniques involving 2D electrophoresis, immuno-detection, and/or proteomics for advanced analysis of SCs.

Investigation of amino acid specificity in the CydX small protein shows sequence plasticity at the functional level.

Small proteins are a new and expanding area of research. Many characterized small proteins are composed of a single hydrophobic α-helix, and the functional requirements of their limited amino acid sequence are not well understood. One hydrophobic small protein, CydX, has been shown to be a component of the cytochrome bd oxidase complex in Escherichia coli, and is required for enzyme function. To investigate small protein sequence specificity, an alanine scanning mutagenesis on the small protein CydX was conducted using mutant alleles expressed from the E. coli chromosome at the wild-type locus. The resulting mutant strains were assayed for CydX function. No single amino acid was required to maintain wild-type resistance to ß-mercaptoethanol. However, substitutions of 10-amino acid blocks indicated that the N-terminus of the protein was required for wild-type CydX activity. A series of double mutants showed that multiple mutations at the N-terminus led to ß-mercaptoethanol sensitivity in vivo. Triple mutants showed both in vivo and in vitro phenotypes. Together, these data provide evidence suggesting a high level of functional plasticity in CydX, in which multiple amino acids may work cooperatively to facilitate CydX function.

Architecture of Human Mitochondrial Respiratory Megacomplex I2III2IV2.

The respiratory megacomplex represents the highest-order assembly of respiratory chain complexes, and it allows mitochondria to respond to energy-requiring conditions. To understand its architecture, we examined the human respiratory chain megacomplex-I2III2IV2 (MCI2III2IV2) with 140 subunits and a subset of associated cofactors using cryo-electron microscopy. The MCI2III2IV2 forms a circular structure with the dimeric CIII located in the center, where it is surrounded by two copies each of CI and CIV. Two cytochrome c (Cyt.c) molecules are positioned to accept electrons on the surface of the c1 state CIII dimer. Analyses indicate that CII could insert into the gaps between CI and CIV to form a closed ring, which we termed the electron transport chain supercomplex. The structure not only reveals the precise assignment of individual subunits of human CI and CIII, but also enables future in-depth analysis of the electron transport chain as a whole.

Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE).

Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in-gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN-PAGE).

Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies.

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site.

Cytochrome bd ubiquinol oxidase uses the electron transport from ubiquinol to oxygen to establish a proton gradient across the membrane. The enzyme complex consists of subunits CydA and B and contains two b- and one d-type hemes as cofactors. Recently, it was proposed that a third subunit named CydX is essential for the function of the complex. Here, we show that CydX is indeed a subunit of purified Escherichia coli cytochrome bd oxidase and that the small protein is needed either for the assembly or the stability of the active site di-heme center and, thus, is essential for oxidase activity.

An investigation into membrane bound redox carriers involved in energy transduction mechanism in Brevibacterium linens DSM 20158 with unsequenced genome.

Brevibacterium linens (B. linens) DSM 20158 with an unsequenced genome can be used as a non-pathogenic model to study features it has in common with other unsequenced pathogens of the same genus on the basis of comparative proteome analysis. The most efficient way to kill a pathogen is to target its energy transduction mechanism. In the present study, we have identified the redox protein complexes involved in the electron transport chain of B. linens DSM 20158 from their clear homology with the shot-gun genome sequenced strain BL2 of B. linens by using the SDS-Polyacrylamide gel electrophoresis coupled with nano LC-MS/MS mass spectrometry. B. linens is found to have a branched electron transport chain (Respiratory chain), in which electrons can enter the respiratory chain either at NADH (Complex I) or at Complex II level or at the cytochrome level. Moreover, we are able to isolate, purify, and characterize the membrane bound Complex II (succinate dehydrogenase), Complex III (menaquinone cytochrome c reductase cytochrome c subunit, Complex IV (cytochrome c oxidase), and Complex V (ATP synthase) of B. linens strain DSM 20158.

Electron transport in acetate-grown Methanosarcina acetivorans.

BACKGROUND: Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H2 as an intermediate, although the great majority of acetotrophs are unable to metabolize H2. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H2-utilizing Methanosarcina acetivorans from H2-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens. RESULTS: A component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans. CONCLUSIONS: The results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H2-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.

The fully oxidized form of the cytochrome bd quinol oxidase from E. coli does not participate in the catalytic cycle: direct evidence from rapid kinetics studies.

Cytochrome bd catalyzes the two-electron oxidation of either ubiquinol or menaquinol and the four-electron reduction of O(2) to H(2)O. In the current work, the rates of reduction of the fully oxidized and oxoferryl forms of the enzyme by the 2-electron donor ubiquinol-1 and single electron donor N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) have been examined by stopped-flow techniques. Reduction of the all-ferric form of the enzyme is 1000-fold slower than required for a step in the catalytic cycle, whereas the observed rates of reduction of the oxoferryl and singly-reduced forms of the cytochrome are consistent with the catalytic turnover. The data support models of the catalytic cycle which do not include the fully oxidized form of the enzyme as an intermediate.

Redox control of fast ligand dissociation from Escherichia coli cytochrome bd.

Bacterial bd-type quinol oxidases, such as cytochrome bd from Escherichia coli, contain three hemes, but no copper. In contrast to heme-copper oxidases and similarly to globins, single electron-reduced cytochrome bd forms stable complexes with O(2), NO and CO at ferrous heme d. Kinetics of ligand dissociation from heme d(2+) in the single electron- and fully-reduced cytochrome bd from E. coli has been investigated by rapid mixing spectrophotometry at 20 degrees C. Data show that (i) O(2) dissociates at 78 s(-1), (ii) NO and CO dissociation is fast as compared to heme-copper oxidases and (iii) dissociation in the single electron-reduced state is hindered as compared to the fully-reduced enzyme. Presumably, rapid ligand dissociation requires reduced heme b(595). As NO, an inhibitor of respiratory oxidases, is involved in the immune response against microbial infection, the rapid dissociation of NO from cytochrome bd may have important bearings on the patho-physiology of enterobacteria.

Assembly of respiratory complexes I, III, and IV into NADH oxidase supercomplex stabilizes complex I in Paracoccus denitrificans.

Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete "respirasome." Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.