Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

The role of glycine residues 140 and 141 of subunit B in the functional ubiquinone binding site of the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

The Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) is the main entrance for electrons into the respiratory chain of many marine and pathogenic bacteria. The enzyme accepts electrons from NADH and donates them to ubiquinone, and the free energy released by this redox reaction is used to create an electrochemical gradient of sodium across the cell membrane. Here we report the role of glycine 140 and glycine 141 of the NqrB subunit in the functional binding of ubiquinone. Mutations at these residues altered the affinity of the enzyme for ubiquinol. Moreover, mutations in residue NqrB-G140 almost completely abolished the electron transfer to ubiquinone. Thus, NqrB-G140 and -G141 are critical for the binding and reaction of Na(+)-NQR with its electron acceptor, ubiquinone.     

Energy Transducing Roles of Antiporter-like Subunits in Escherichia coli NDH-1 with Main Focus on Subunit NuoN (ND2)

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (_NLys³⁹⁵) is conserved in NuoL (_LLys³⁹⁹) but is replaced by glutamic acid (_MGlu⁴⁰⁷) in NuoM. Our mutation study on _NLys³⁹⁵ suggests that this residue participates in the proton translocation. Furthermore, we found that _MGlu⁴⁰⁷ is also essential and most likely interacts with conserved _LArg¹⁷⁵. Glutamic acids, _NGlu¹³³, _MGlu¹⁴⁴, and _LGlu¹⁴⁴, are corresponding residues. Unlike mutants of _MGlu¹⁴⁴ and _LGlu¹⁴⁴, mutation of _NGlu¹³³ scarcely affected the energy-transducing activities. However, a double mutant of _NGlu¹³³ and nearby _KGlu⁷² showed significant inhibition of these activities. This suggests that _NGlu¹³³ bears a functional role similar to _LGlu¹⁴⁴ and _MGlu¹⁴⁴ but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that _NLys¹⁵⁸ is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of _NTM14 interacts with the _Mβ sheet located on the opposite side of helix HL. Taken together, the mechanism of H⁺ translocation in NDH-1 is discussed.     

Post-translational Modifications near the Quinone Binding Site of Mammalian Complex I

Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N^G and ω-N^G′ nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.     

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (Etf_Af) and butyryl-CoA dehydrogenase (Bcd_Af) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. Etf_Af contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The Etf_Af-NAD⁺ complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH⁻ is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH⁻ by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD^⨪, which donates this electron further to Dh-FAD of Bcd_Af after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH^•, immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH⁻ that converts crotonyl-CoA to butyryl-CoA.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Modulation of the Respiratory Supercomplexes in Yeast: ENHANCED FORMATION OF CYTOCHROME OXIDASE INCREASES THE STABILITY AND ABUNDANCE OF RESPIRATORY SUPERCOMPLEXES*

Yeast cells deficient in the Rieske iron-sulfur subunit (Rip1) of ubiquinol-cytochrome c reductase (bc₁) accumulate a late core assembly intermediate, which weakly associates with cytochrome oxidase (CcO) in a respiratory supercomplex. Expression of the N-terminal half of Rip1, which lacks the C-terminal FeS-containing globular domain (designated N-Rip1), results in a marked stabilization of trimeric and tetrameric bc₁-CcO supercomplexes. Another bc₁ mutant (qcr9Δ) stalled at the same assembly intermediate is likewise converted to stable supercomplex species by the expression of N-Rip1, but not by expression of intact Rip1. The N-Rip1-induced stabilization of bc₁-CcO supercomplexes is independent of the Bcs1 translocase, which mediates Rip1 translocation during bc₁ biogenesis. N-Rip1 induces the stabilization of bc₁-CcO supercomplexes through an enhanced formation of CcO. The association of N-Rip1 with the late core bc₁ assembly intermediate appears to confer stabilization of a CcO assembly intermediate. This induced stabilization of CcO is dependent on the Rcf1 supercomplex stabilization factor and only partially dependent on the presence of cardiolipin. N-Rip1 exerts a related induction of CcO stabilization in WT yeast, resulting in enhanced respiration. Additionally, the impact of CcO stabilization on supercomplexes was observed by means other than expression of N-Rip1 (via overexpression of CcO subunits Cox4 and Cox5a), demonstrating that this is a general phenomenon. This study presents the first evidence showing that supercomplexes can be stabilized by the stimulated formation of CcO.     

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.     

Soluble,Prefibrillar α-Synuclein Oligomers Promote Complex I-dependent,Ca2+-induced Mitochondrial Dysfunction

α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca²⁺ oscillations that burden mitochondria, we examined mitochondrial Ca²⁺ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca²⁺, promoted Ca²⁺-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca²⁺. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca²⁺ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca²⁺-handling requirements.     

The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na⁺-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD⁺ with the generation of a primary electrochemical Na⁺ potential across its cytoplasmic membrane. In previous assays in which Ti³⁺ was used to reduce ferredoxin, Na⁺ transport was observed, but not a Na⁺ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD⁺ reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na⁺ dependence of this electron transfer reaction. The K_m was 0.2 mm. Na⁺ could be partly substituted by Li⁺. Na⁺ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na⁺ as a coupling ion. Electron transport from reduced ferredoxin to NAD⁺ was coupled to electrogenic Na⁺ transport, indicating the generation of Δ$\tilde{\mu }$_Na⁺. Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of Δ$\tilde{\mu }$_Na⁺, and was accompanied by Na⁺ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na⁺ gradient. The physiological significance of this finding is discussed.     

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q_D) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q_D site (MSQ_D) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with ¹H₂O/²H₂O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ_D binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in ¹H₂O and ²H₂O samples and by selective detection of the exchanged deuterons using Q-band ²H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b_D. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q_D site are discussed, in light of the unusually high thermodynamic stability of MSQ_D.     

Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.     

NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His⁴⁹ both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E_m value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the Mo-bisPGD cofactor. To demonstrate that decreasing the FS0 cluster E_m results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg⁹⁴) in the vicinity of FS0 to a Ser residue. In this case, the E_m of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to ∼30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdo-NarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, ⁴⁹HGVNCTG⁵⁵, must be correctly positioned to ensure holoenzyme maturation.     

Roles of Subunit NuoK (ND4L) in the Energy-transducing Mechanism of Escherichia coli NDH-1 (NADH:Quinone Oxidoreductase)

The bacterial H⁺-translocating NADH:quinone oxidoreductase (NDH-1) catalyzes electron transfer from NADH to quinone coupled with proton pumping across the cytoplasmic membrane. The NuoK subunit (counterpart of the mitochondrial ND4L subunit) is one of the seven hydrophobic subunits in the membrane domain and bears three transmembrane segments (TM1–3). Two glutamic residues located in the adjacent transmembrane helices of NuoK are important for the energy coupled activity of NDH-1. In particular, mutation of the highly conserved carboxyl residue (_KGlu-36 in TM2) to Ala led to a complete loss of the NDH-1 activities. Mutation of the second conserved carboxyl residue (_KGlu-72 in TM3) moderately reduced the activities. To clarify the contribution of NuoK to the mechanism of proton translocation, we relocated these two conserved residues. When we shifted _KGlu-36 along TM2 to positions 32, 38, 39, and 40, the mutants largely retained energy transducing NDH-1 activities. According to the recent structural information, these positions are located in the vicinity of _KGlu-36, present in the same helix phase, in an immediately before and after helix turn. In an earlier study, a double mutation of two arginine residues located in a short cytoplasmic loop between TM1 and TM2 (loop-1) showed a drastic effect on energy transducing activities. Therefore, the importance of this cytosolic loop of NuoK (_KArg-25, _KArg-26, and _KAsn-27) for the energy transducing activities was extensively studied. The probable roles of subunit NuoK in the energy transducing mechanism of NDH-1 are discussed.     

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.     

Structure Determination and Improved Model of Plant Photosystem I

Photosystem I functions as a sunlight energy converter, catalyzing one of the initial steps in driving oxygenic photosynthesis in cyanobacteria, algae, and higher plants. Functionally, Photosystem I captures sunlight and transfers the excitation energy through an intricate and precisely organized antenna system, consisting of a pigment network, to the center of the molecule, where it is used in the transmembrane electron transfer reaction. Our current understanding of the sophisticated mechanisms underlying these processes has profited greatly from elucidation of the crystal structures of the Photosystem I complex. In this report, we describe the developments that ultimately led to enhanced structural information of plant Photosystem I. In addition, we report an improved crystallographic model at 3.3-Å resolution, which allows analysis of the structure in more detail. An improved electron density map yielded identification and tracing of subunit PsaK. The location of an additional ten β-carotenes as well as five chlorophylls and several loop regions, which were previously uninterpretable, are now modeled. This represents the most complete plant Photosystem I structure obtained thus far, revealing the locations of and interactions among 17 protein subunits and 193 non-covalently bound photochemical cofactors. Using the new crystal structure, we examine the network of contacts among the protein subunits from the structural perspective, which provide the basis for elucidating the functional organization of the complex.     

Protein carbonylation and adipocyte mitochondrial function

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.     

Evolution of respiratory complex I: "supernumerary" subunits are present in the alpha-proteobacterial enzyme

Modern α-proteobacteria are thought to be closely related to the ancient symbiont of eukaryotes, an ancestor of mitochondria. Respiratory complex I from α-proteobacteria and mitochondria is well conserved at the level of the 14 "core" subunits, consistent with that notion. Mitochondrial complex I contains the core subunits, present in all species, and up to 31 "supernumerary" subunits, generally thought to have originated only within eukaryotic lineages. However, the full protein composition of an α-proteobacterial complex I has not been established previously. Here, we report the first purification and characterization of complex I from the α-proteobacterium Paracoccus denitrificans. Single particle electron microscopy shows that the complex has a well defined L-shape. Unexpectedly, in addition to the 14 core subunits, the enzyme also contains homologues of three supernumerary mitochondrial subunits as follows: B17.2, AQDQ/18, and 13 kDa (bovine nomenclature). This finding suggests that evolution of complex I via addition of supernumerary or "accessory" subunits started before the original endosymbiotic event that led to the creation of the eukaryotic cell. It also provides further confirmation that α-proteobacteria are the closest extant relatives of mitochondria.