Iron-sulfur (FeS) proteins are ancient and fundamental to life, being involved in electron transfer and CO
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 Fe
/Fe
and S
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,
Fe-Mössbauer spectroscopy and
H-NMR. Cyclic voltammetry shows that the clusters are redox-active. Our findings reveal that the structures responsible for biological electron transfer and CO
reduction could have formed spontaneously from monomers at the origin of life.
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 CIV5Bcontaining 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.
Abstract
Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane
1
. 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–III
2
–IV
2
supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.
Vesicles formed from single-chain amphiphiles (SCAs) such as fatty acids probably played an important role in the origin of life. A major criticism of the hypothesis that life arose in an early ocean ...hydrothermal environment is that hot temperatures, large pH gradients, high salinity and abundant divalent cations should preclude vesicle formation. However, these arguments are based on model vesicles using 1-3 SCAs, even though Fischer-Tropsch-type synthesis under hydrothermal conditions produces a wide array of fatty acids and 1-alkanols, including abundant C
-C
compounds. Here, we show that mixtures of these C
-C
SCAs form vesicles in aqueous solutions between pH ~6.5 and >12 at modern seawater concentrations of NaCl, Mg
and Ca
. Adding C
isoprenoids improves vesicle stability even further. Vesicles form most readily at temperatures of ~70 °C and require salinity and strongly alkaline conditions to self-assemble. Thus, alkaline hydrothermal conditions not only permit protocell formation at the origin of life but actively favour it.
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.
The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric ...channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas. Possible control of function by dielectric channels and their evolutionary relation to proton channels is explored.
In the present chapter, the structures and mechanisms of the major components of mammalian mitochondrial respiratory chains are reviewed. Particular emphasis is placed on the four protein complexes ...and their cofactors that catalyse the electron transfer pathway between oxidation of NADH and succinate and the reduction of oxygen to water. Current ideas are reviewed of how these electron transfer reactions are coupled to formation of the proton and charge gradient across the inner mitochondrial membrane that is used to drive ATP synthesis. Additional respiratory components that are found in mammalian and plant, fungal and algal mitochondria are also reviewed.
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.
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.
•Yeast monomeric complex IV was isolated with maltosides and stabilised in amphipols.•Its structure was solved at 3.9 Å using cryo-EM and a 178 kDa 3D-model was built.•It includes Cox1–9 but lacks Cox12, Cox13 and Cox26; no Rcf protein was identified.•No large conformational change of CIV is associated with supercomplex formation.
In cytochrome c oxidase (CytcO) reduction of O
to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped ...across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than through the D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D (Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO with O
. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and presumably also for proton pumping.
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