ABSTRACT
Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III2-IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
Subject(s)
Cryoelectron Microscopy , Electron Transport Complex III , Electron Transport Complex II , Electron Transport Complex IV , Electron Transport Complex I , Mitochondria , Mitochondrial Membranes , Electron Transport , Electron Transport Complex III/chemistry , Electron Transport Complex III/metabolism , Electron Transport Complex III/ultrastructure , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Electron Transport Complex IV/ultrastructure , Mitochondria/chemistry , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondria/ultrastructure , Mitochondrial Membranes/chemistry , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/ultrastructure , Electron Transport Complex II/chemistry , Electron Transport Complex II/metabolism , Electron Transport Complex II/ultrastructure , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Electron Transport Complex I/ultrastructure , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Molecular Dynamics Simulation , Binding Sites , Evolution, MolecularABSTRACT
The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII-CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.
Subject(s)
Electron Transport Complex III/metabolism , Electron Transport Complex IV/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Cryoelectron Microscopy/methods , Electron Transport Complex III/chemistry , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/physiology , Hypoxia/metabolism , Mitochondria/chemistry , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Protein Isoforms , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e- stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO's hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.
Subject(s)
Electron Transport Complex IV/chemistry , Heme/chemistry , Oxidoreductases/chemistry , Bacteria/metabolism , Copper/chemistry , Copper/metabolism , Electron Transport Complex IV/genetics , Electron Transport Complex IV/metabolism , Ion Transport , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Oxidation-Reduction , Oxidoreductases/metabolism , Oxygen/metabolism , Proton Pumps/metabolism , Protons , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules. The additional redox-sensitive band reported at 1736â¯cm-1 in bovine CcO and previously assigned to D51 is absent from yeast CcO and couldn't be restored by introduction of a D residue at the equivalent position of the yeast protein. Redox spectra of yeast CcO also show much smaller changes in the amide I region, which may relate to structural differences in the region around D51 and the subunit I/II interface.
Subject(s)
Carbon Monoxide/metabolism , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Mutation , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Spectroscopy, Fourier Transform Infrared/methods , Animals , Catalytic Domain , Cattle , Electron Transport Complex IV/genetics , Kinetics , Light , Oxidation-Reduction , Photolysis , Protein Conformation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3'-2' phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications. We established routes for the chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks, and we demonstrated that DNA duplexes were destabilized upon replacement with tPhoNA. We engineered a novel tPhoNA synthetase enzyme and, with a previously reported XNA reverse transcriptase, demonstrated that tPhoNA is a viable genetic material (with an aggregate error rate of approximately 17 × 10-3 per base) compatible with the isolation of functional XNAs. In vivo experiments to test tPhoNA orthogonality showed that the E. coli cellular machinery had only very limited potential to access genetic information in tPhoNA. Our work is the first report of a synthetic genetic material modified in both sugar and phosphate backbone moieties and represents a significant advance in biorthogonality toward the introduction of XNA systems in vivo.
Subject(s)
DNA/chemistry , Organophosphonates/chemistry , Polymers/metabolism , Xenobiotics/metabolism , DNA/metabolism , Ligases/chemistry , Ligases/metabolism , Models, Molecular , Molecular Structure , Organophosphonates/metabolism , Polymers/chemistry , Protein Engineering , Xenobiotics/chemistryABSTRACT
The activation of the peroxynitrite anion (PN) by hemoproteins, which leads to its detoxification or, on the contrary to the enhancement of its cytotoxic activity, is a reaction of physiological importance that is still poorly understood. It has been known for some years that the reaction of hemoproteins, notably cytochrome P450, with PN leads to the buildup of an intermediate species with a Soret band at â¼435 nm (I435). The nature of this intermediate is, however, debated. On the one hand, I435 has been presented as a compound II species that can be photoactivated to compound I. A competing alternative involves the assignment of I435 to a ferric-nitrosyl species. Similar to cytochromes P450, the buildup of I435 occurs in nitric oxide synthases (NOSs) upon their reaction with excess PN. Interestingly, the NOS isoforms vary in their capacity to detoxify/activate PN, although they all show the buildup of I435. To better understand PN activation/detoxification by heme proteins, a definitive assignment of I435 is needed. Here we used a combination of fine kinetic analysis under specific conditions (pH, PN concentrations, and PN/NOSs ratios) to probe the formation of I435. These studies revealed that I435 is not formed upon homolytic cleavage of the O-O bond of PN, but instead arises from side reactions associated with excess PN. Characterization of I435 by resonance Raman spectroscopy allowed its identification as a ferric iron-nitrosyl complex. Our study indicates that the model used so far to depict PN interactions with hemo-thiolate proteins, i.e., leading to the formation and accumulation of compound II, needs to be reconsidered.
Subject(s)
Nitric Oxide Synthase/metabolism , Peroxynitrous Acid/metabolism , Hemeproteins/metabolism , Hydrogen-Ion Concentration , Kinetics , Staphylococcus aureus/enzymologyABSTRACT
We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.4ms) than with the bovine oxidase (~1ms) and a larger fraction CuA was oxidized with the yeast than with the bovine oxidase in the peroxy (PR) to F reaction. Furthermore, upon replacement of Glu243, located at the end of the so-called D proton pathway, by Asp the PRâF and FâO reactions were slowed by factors of ~3 and ~10, respectively, and electron transfer from CuA to heme a during the PRâF reaction was not observed. These data indicate that during reduction of dioxygen protons are transferred through the D pathway, via Glu243, to the catalytic site in the yeast mitochondrial oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
Subject(s)
Electron Transport Complex IV/metabolism , Electron Transport , Mutation, Missense , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Molecular Sequence Data , Oxidation-Reduction , Oxygen/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.
Subject(s)
Electron Transport Complex IV/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Water/chemistry , Animals , Catalysis , Cattle , Ferrous Compounds , Hydrogen Bonding , Oxidation-Reduction , PhotolysisABSTRACT
The polypeptide hormone Amylin (also known as islet amyloid polypeptide) plays a role in regulation of glucose metabolism, but forms pancreatic islet amyloid deposits in type 2 diabetes. The process of islet amyloid formation contributes to ß-cell dysfunction and the development of the disease. Amylin is produced as a pro-from and undergoes processing prior to secretion. The mature hormone contains an amidated C-terminus. Analysis of an alignment of vertebrate amylin sequences reveals that the processing signal for amidation is strictly conserved. Furthermore, the enzyme responsible for C-terminal amidation is found in all of these organisms. Comparison of the physiologically relevant amidated form to a variant with a free C-terminus (Amylin-COO-) shows that replacement of the C-terminal amide with a carboxylate slows, but does not prevent amyloid formation. Pre-fibrillar species produced by both variants are toxic to cultured ß-cells, although hAmylin-COO- is moderately less so. Amyloid fibrils produced by either peptide are not toxic. Prior work (ACS Pharmacol. Translational. Sci. 1, 132-49 (2018)) shows that Amylin- COO- exhibits a 58-fold reduction in activation of the Amylin1 receptor and 20-fold reduction in activation of the Amylin3 receptor. Thus, hAmylin-COO- exhibits significant toxicity, but significantly reduced activity and offers a reagent for studies which aim to decouple hAmylin's toxic effects from its activity. The different behaviours of free and C-terminal amidated Amylin should be considered when designing systems to produce the polypeptide recombinantly.
Subject(s)
Diabetes Mellitus, Type 2 , Peptide Hormones , Humans , Islet Amyloid Polypeptide/chemistry , Diabetes Mellitus, Type 2/metabolism , Amides , Amyloidogenic Proteins , Amyloid/chemistryABSTRACT
Point mutations of E243D and I67N were introduced into subunit I of a 6histidine-tagged (6H-WT) form of yeast Saccharomyces cerevisiae mitochondrial cytochrome c oxidase. The two mutants (6H-E243D(I) and 6H-I67N(I)) were purified and showed ≈50 and 10% of the 6H-WT turnover number. Light-induced CO photolysis FTIR difference spectra of the 6H-WT showed a peak/trough at 1749/1740cm(-1), as seen in bovine CcO, which downshifted by 7cm(-1) in D(2)O. The bands shifted to 1736/1762cm(-1) in 6H-E243D(I), establishing that the carboxyl group affected by CO binding in mitochondrial CcOs is E243. In 6H-I67N(I), the trough at 1740cm(-1) was shifted to 1743cm(-1) and its accompanying peak intensity was greatly reduced. This confirms that the I67N mutation interferes with conformational alterations around E243. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Subject(s)
Electron Transport Complex IV/metabolism , Mitochondrial Proteins/metabolism , Mutation, Missense , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Substitution , Animals , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Cattle , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Photolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed.
Subject(s)
Electron Transport Complex IV/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Animals , Cattle , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/genetics , Heme/chemistry , Heme/metabolism , Mitochondria/enzymology , Mitochondria/metabolism , Models, Molecular , Mutation , Oxidoreductases/chemistry , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the binding of Na(+) and Ca(2+)cations to bovine cytochrome c oxidase in its fully oxidized and partially reduced, cyanide-ligated (a(2+)a3(3+)-CN) (mixed valence) forms. These ions induced distinctly different IR binding spectra, indicating that the induced structural changes are different. Despite this, their binding spectra were mutually exclusive, confirming their known competitive binding behavior. Dissociation constants for Na(+) and Ca(2+) with the oxidized enzyme were 1.2 mM and 11 µM, respectively and Na(+) binding appeared to involve cooperative binding of two Na(+). Ca(2+) binding induced a large IR spectrum, with prominent amide I/II polypeptide changes, bandshifts assigned to carboxylate and an arginine, and a number of bandshifts of heme a. The Na(+)-induced binding spectrum showed much weaker amide I/II and heme a changes but had similar shifts assignable to carboxylate and arginine residues. Yeast CcO also displayed a calcium-induced IR and UV/visible binding spectra, though of lower intensities. This was attributed to the difficulty in fully depleting Ca(2+) from its binding site, as has been found with bacterial CcOs. The implications of Ca(2+)/Na(+) ion binding are discussed in terms of structure and possible modulation of core catalytic function.
Subject(s)
Calcium/metabolism , Calcium/pharmacology , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Sodium/metabolism , Sodium/pharmacology , Spectroscopy, Fourier Transform Infrared , Animals , Cattle , Cyanides/metabolism , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein Conformation/drug effects , Saccharomyces cerevisiae/enzymologyABSTRACT
Nitric oxide is produced in mammals by a class of enzymes called NO synthases (NOSs). It plays a central role in cellular signalling but also has deleterious effects, as it leads to the production of reactive oxygen and nitrogen species. NO forms a relatively stable adduct with ferrous haem proteins, which, in the case of NOS, is also a key catalytic intermediate. Despite extensive studies on the ferrous nitrosyl complex of other haem proteins (in particular myoglobin), little characterisation has been performed in the case of NOS. We report here a temperature-dependent EPR study of the ferrous nitrosyl complex of the inducible mammalian NOS and the bacterial NOS-like protein from Bacillus subtilis. The results show that the overall behaviours are similar to those observed for other haem proteins, but with distinct ratios between axial and rhombic forms in the case of the two NOS proteins. The distal environment appears to control the existence of the axial form and the evolution of the rhombic form.
Subject(s)
Coordination Complexes/chemistry , Electron Spin Resonance Spectroscopy , Ferrous Compounds/chemistry , Nitric Oxide Synthase Type II/chemistry , Nitric Oxide/chemistry , Bacillus subtilis/enzymology , Isoenzymes/chemistry , Isoenzymes/metabolism , Myoglobin/chemistry , Myoglobin/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Substrate Specificity , TemperatureABSTRACT
Assignments of IR bands of reduced minus oxidized IR difference spectra of bovine and related cytochrome c oxidases are reviewed and their linkages to specific metal centres are assessed. To aid this, redox-poised difference spectra in the presence of cyanide or carbon monoxide are presented. These ligands fix the redox states of either haem a3 alone or haem a3 and CuB respectively, while allowing redox cycling of the remaining centres.
Subject(s)
Carbon Monoxide/chemistry , Electron Transport Complex IV/chemistry , Heme/analogs & derivatives , Animals , Carbon Monoxide/metabolism , Cattle , Copper/chemistry , Cyanides/chemistry , Heme/chemistry , Infrared Rays , Ligands , Oxidation-Reduction/radiation effects , Spectroscopy, Fourier Transform InfraredABSTRACT
Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.
Subject(s)
Electron Transport Complex IV/genetics , Histidine/genetics , Mitochondria/enzymology , Mitochondria/genetics , Point Mutation/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Electron Transport Complex IV/isolation & purification , Nuclear Proteins/chemical synthesis , Nuclear Proteins/isolation & purification , Protein Subunits/chemical synthesis , Protein Subunits/isolation & purification , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/isolation & purificationABSTRACT
In mitochondria, complex IV (CIV) can be found as a monomer, a dimer or in association with other respiratory complexes. The atomic structure of the yeast S. cerevisiae CIV in a supercomplex (SC) with complex III (CIII) pointed to a region of significant conformational changes compared to the homologous mammalian CIV structures. These changes involved the matrix side domain of Cox5A at the CIII-CIV interface, and it was suggested that it could be required for SC formation. To investigate this, we solved the structure of the isolated monomeric CIV from S. cerevisiae stabilised in amphipol A8-35 at 3.9 Å using cryo-electron microscopy. Only a minor change in flexibility was seen in this Cox5A region, ruling out large CIV conformational shift for interaction with CIII and confirming the different fold of the yeast Cox5A subunit compared to mammalian homologues. Other differences in structure were the absence of two canonical subunits, Cox12 and Cox13, as well as Cox26, which is unique to the yeast CIV. Their absence is most likely due to the protein purification protocol used to isolate CIV from the III-IV SC.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Animals , Cryoelectron Microscopy/methods , Electron Transport Complex IV/metabolism , Mammals/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
In 2005, it was found that the fluorescence of crystals of the major light-harvesting complex LHCII of green plants is significantly quenched when compared to the fluorescence of isolated LHCII (A. A. Pascal et al., Nature, 2005, 436, 134-137). The Raman spectrum of crystallized LHCII was also found to be different from that of isolated LHCII but very similar to that of aggregated LHCII, which has often been considered a good model system for studying nonphotochemical quenching (NPQ), the major protection mechanism of plants against photodamage in high light. It was proposed that in the crystal LHCII adopts a similar (quenching) conformation as during NPQ and indeed similar changes in the Raman spectrum were observed during NPQ in vivo (A. V. Ruban et al., Nature, 2007, 450, 575-579). We now compared the fluorescence of various types of crystals, differing in morphology and age. Each type gave rise to its own characteristic mono-exponential fluorescence lifetime, which was 5 to 10 times shorter than that of isolated LHCII. This indicates that fluorescence is not quenched by random impurities and packing defects (as proposed recently by T. Barros et al., EMBO Journal, 2009, 28, 298-306), but that LHCII adopts a particular structure in each crystal type, that leads to fluorescence quenching. Most interestingly, the extent of quenching appears to depend on the crystal morphology, indicating that also the crystal structure depends on this crystal morphology but at the moment no data are available to correlate the crystals' structural changes to changes in fluorescence lifetime.
Subject(s)
Light-Harvesting Protein Complexes/chemistry , Crystallization , Microscopy, Fluorescence , Spectrum Analysis, RamanABSTRACT
Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO2 fixation. FeS clusters have structures similar to the unit-cell of FeS minerals such as greigite, found in hydrothermal systems linked with the origin of life. However, the prebiotic pathway from mineral surfaces to biological clusters is unknown. Here we show that FeS clusters form spontaneously through interactions of inorganic Fe2+/Fe3+ and S2- with micromolar concentrations of the amino acid cysteine in water at alkaline pH. Bicarbonate ions stabilize the clusters and even promote cluster formation alone at concentrations >10 mM, probably through salting-out effects. We demonstrate robust, concentration-dependent formation of [4Fe4S], [2Fe2S] and mononuclear iron clusters using UV-Vis spectroscopy, 57Fe-Mössbauer spectroscopy and 1H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO2 reduction could have formed spontaneously from monomers at the origin of life.
Subject(s)
Cysteine/chemistry , Iron/chemistry , Models, Chemical , Origin of Life , Sulfides/chemistry , Sulfur/chemistry , Bicarbonates/chemistry , Carbon Dioxide/chemistry , Electrochemical Techniques , Electron Transport , Hydrogen-Ion Concentration , Oxidation-Reduction , Spectroscopy, MossbauerABSTRACT
Despite the fact that ferrous myeloperoxidase (MPO) can bind both O(2) and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous-CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, k(on) and k(off) were 0.14 mM(-1) x s(-1) and 0.23 s(-1), respectively, yielding an unusually high K(D) of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O(2). The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pK(a) of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm(-1). Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pK(a) approximately 7.4) with the 1927 cm(-1) form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.
Subject(s)
Carbon Monoxide/metabolism , Peroxidase/metabolism , Catalytic Domain , Heme/metabolism , Humans , Iron/metabolism , Ligands , Models, Molecular , Oxygen/metabolism , Peroxidase/chemistry , Protein Binding , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Substrate SpecificityABSTRACT
Electrochemistry coupled with Fourier transform infrared (IR) spectroscopy was used to investigate the redox properties of recombinant alternative ubiquinol oxidase from Trypanosoma brucei, the organism responsible for African sleeping sickness. Stepwise reduction of the fully oxidized resting state of recombinant alternative ubiquinol oxidase revealed two distinct IR redox difference spectra. The first of these, signal 1, titrates in the reductive direction as an n = 2 Nernstian component with an apparent midpoint potential of 80 mV at pH 7.0. However, reoxidation of signal 1 in the same potential range under anaerobic conditions did not occur and only began with potentials in excess of 500 mV. Reoxidation by introduction of oxygen was also unsuccessful. Signal 1 contained clear features that can be assigned to protonation of at least one carboxylate group, further perturbations of carboxylic and histidine residues, bound ubiquinone, and a negative band at 1554 cm(-1) that might arise from a radical in the fully oxidized protein. A second distinct IR redox difference spectrum, signal 2, appeared more slowly once signal 1 had been reduced. This component could be reoxidized with potentials above 100 mV. In addition, when both signals 1 and 2 were reduced, introduction of oxygen caused rapid oxidation of both components. These data are interpreted in terms of the possible active site structure and mechanism of oxygen reduction to water.