Structural and functional mechanisms of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in mitochondria. It catalyzes the four-electron reduction of O2 to H2O and harnesses the redox energy to drive unidirectional proton translocation against a proton electrochemical gradient. A great deal of research has been conducted to comprehend the molecular properties of CcO. However, the mechanism by which the oxygen reduction reaction is coupled to proton translocation remains poorly understood. Here, we review the chemical properties of a variety of key oxygen intermediates of bovine CcO (bCcO) revealed by time-resolved resonance Raman spectroscopy and the structural features of the enzyme uncovered by serial femtosecond crystallography, an innovative technique that allows structural determination at room temperature without radiation damage. The implications of these data on the proton translocation mechanism are discussed.

ATAD3 is a limiting factor in mitochondrial biogenesis and adipogenesis of white adipocyte-like 3T3-L1 cells.

ATAD3 is a vital ATPase of the inner mitochondrial membrane of pluri-cellular eukaryotes, with largely unknown functions but early required for organism development as necessary for mitochondrial biogenesis. ATAD3 knock-down in C. elegans inhibits at first the development of adipocyte-like intestinal tissue so we used mouse adipocyte model 3T3-L1 cells to analyze ATAD3 functions during adipogenesis and lipogenesis in a mammalian model. ATAD3 function was studied by stable and transient modulation of ATAD3 expression in adipogenesis- induced 3T3-L1 cells using Knock-Down and overexpression strategies, exploring different steps of adipocyte differentiation and lipogenesis. We show that (i) an increase in ATAD3 is preceding differentiation-induced mitochondrial biogenesis; (ii) downregulation of ATAD3 inhibits adipogenesis, lipogenesis, and impedes overexpression of many mitochondrial proteins; (iii) ATAD3 re-expression rescues the phenotype of ATAD3 KD, and (iv) differentiation and lipogenesis are accelerated by ATAD3 overexpression, but inhibited by expression of a dominant-negative mutant. We further show that the ATAD3 KD phenotype is not due to altered insulin signal but involves a limitation of mitochondrial biogenesis linked to Drp1. These results demonstrate that ATAD3 is limiting for in vitro mitochondrial biogenesis and adipogenesis/lipogenesis and therefore that ATAD3 mutation/over- or under-expression could be involved in adipogenic and lipogenic pathologies.

A reliable benchmark of the last 640,000 years millennial climate variability.

How often have past climates undergone abrupt transitions? While our understanding of millennial variability during the past 130,000 years is well established, with precise dates available, such information on previous climate cycles is limited. To address this question, we identified 196 abrupt transitions in the δ18O record of the well-dated Chinese composite speleothem for the last 640,000 years. These results correspond to abrupt changes in the strength of the East Asian Monsoon, which align with the Greenland stadials and interstadials observed in the North Atlantic region during the last 130,000 years before present. These precise dates of past abrupt climate changes constitute a reliable and necessary benchmark for Earth System models used to study future climate scenarios.

Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

A punctuated equilibrium analysis of the climate evolution of cenozoic exhibits a hierarchy of abrupt transitions.

The Earth's climate has experienced numerous critical transitions during its history, which have often been accompanied by massive and rapid changes in the biosphere. Such transitions are evidenced in various proxy records covering different timescales. The goal is then to identify, date, characterize, and rank past critical transitions in terms of importance, thus possibly yielding a more thorough perspective on climatic history. To illustrate such an approach, which is inspired by the punctuated equilibrium perspective on the theory of evolution, we have analyzed 2 key high-resolution datasets: the CENOGRID marine compilation (past 66 Myr), and North Atlantic U1308 record (past 3.3 Myr). By combining recurrence analysis of the individual time series with a multivariate representation of the system based on the theory of the quasi-potential, we identify the key abrupt transitions associated with major regime changes that separate various clusters of climate variability. This allows interpreting the time-evolution of the system as a trajectory taking place in a dynamical landscape, whose multiscale features describe a hierarchy of metastable states and associated tipping points.

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu B ) and a heme a 3 iron, where O 2 binds and is reduced to water. CO is a versatile O 2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (Cu B -CO), where CO is bound to Cu B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the Cu B and the heme a 3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

The PaleoJump database for abrupt transitions in past climates.

Tipping points (TPs) in Earth's climate system have been the subject of increasing interest and concern in recent years, given the risk that anthropogenic forcing could cause abrupt, potentially irreversible, climate transitions. Paleoclimate records are essential for identifying past TPs and for gaining a thorough understanding of the underlying nonlinearities and bifurcation mechanisms. However, the quality, resolution, and reliability of these records can vary, making it important to carefully select the ones that provide the most accurate representation of past climates. Moreover, as paleoclimate time series vary in their origin, time spans, and periodicities, an objective, automated methodology is crucial for identifying and comparing TPs. To address these challenges, we introduce the open-source PaleoJump database, which contains a collection of carefully selected, high-resolution records originating in ice cores, marine sediments, speleothems, terrestrial records, and lake sediments. These records describe climate variability on centennial, millennial and longer time scales and cover all the continents and ocean basins. We provide an overview of their spatial distribution and discuss the gaps in coverage. Our statistical methodology includes an augmented Kolmogorov-Smirnov test and Recurrence Quantification Analysis; it is applied here, for illustration purposes, to selected records in which abrupt transitions are automatically detected and the presence of potential tipping elements is investigated. These transitions are shown in the PaleoJump database along with other essential information about the records, including location, temporal scale and resolution, as well as temporal plots. This open-source database represents, therefore, a valuable resource for researchers investigating TPs in past climates.

Structural basis for functional properties of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, CuB and heme a3, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand-binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation. However, metal centers in proteins can be susceptible to X-ray-induced radiation damage, raising questions about the reliability of conclusions drawn from these studies. Here, we used microspectroscopy-coupled X-ray crystallography to interrogate how the structural integrity of bovine CcO in the fully oxidized state (O) is modulated by synchrotron radiation. Spectroscopic data showed that, upon X-ray exposure, O was converted to a hybrid O∗ state where all the four metal centers were reduced, but the protein matrix was trapped in the genuine O conformation and the ligands in the BNC remained intact. Annealing the O∗ crystal above the glass transition temperature induced relaxation of the O∗ structure to a new R∗ structure, wherein the protein matrix converted to the fully reduced R conformation with the exception of helix X, which partly remained in the O conformation because of incomplete dissociation of the ligands from the BNC. We conclude from these data that reevaluation of reported CcO structures obtained with synchrotron light sources is merited.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Ocean circulation, ice shelf, and sea ice interactions explain Dansgaard-Oeschger cycles.

The last glacial interval experienced abrupt climatic changes called Dansgaard-Oeschger (DO) events. These events manifest themselves as rapid increases followed by slow decreases of oxygen isotope ratios in Greenland ice core records. Despite promising advances, a comprehensive theory of the DO cycles, with their repeated ups and downs of isotope ratios, is still lacking. Here, based on earlier hypotheses, we introduce a dynamical model that explains the DO variability by rapid retreat and slow regrowth of thick ice shelves and thin sea ice in conjunction with changing subsurface water temperatures due to insulation by the ice cover. Our model successfully reproduces observed features of the records, such as the sawtooth shape of the DO cycles, waiting times between DO events across the last glacial, and the shifted antiphase relationship between Greenland and Antarctic ice cores. Our results show that these features can be obtained via internal feedbacks alone. Warming subsurface waters could have also contributed to the triggering of Heinrich events. Our model thus offers a unified framework for explaining major features of multimillennial climate variability during glacial intervals.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

The impact of Last Glacial climate variability in west-European loess revealed by radiocarbon dating of fossil earthworm granules.

The characterization of Last Glacial millennial-timescale warming phases, known as interstadials or Dansgaard-Oeschger events, requires precise chronologies for the study of paleoclimate records. On the European continent, such chronologies are only available for several Last Glacial pollen and rare speleothem archives principally located in the Mediterranean domain. Farther north, in continental lowlands, numerous high-resolution records of loess and paleosols sequences show a consistent environmental response to stadial-interstadial cycles. However, the limited precision and accuracy of luminescence dating methods commonly used in loess deposits preclude exact correlations of paleosol horizons with Greenland interstadials. To overcome this problem, a radiocarbon dating protocol has been developed to date earthworm calcite granules from the reference loess sequence of Nussloch (Germany). Its application yields a consistent radiocarbon chronology of all soil horizons formed between 47 and 20 ka and unambiguously shows the correlation of every Greenland interstadial identified in isotope records with specific soil horizons. Furthermore, eight additional minor soil horizons dated between 27.5 and 21 ka only correlate with minor decreases in Greenland dust records. This dating strategy reveals the high sensitivity of loess paleoenvironments to Northern Hemisphere climate changes. A connection between loess sedimentation rate, Fennoscandian ice sheet dynamics, and sea level changes is proposed. The chronological improvements enabled by the radiocarbon "earthworm clock" thus strongly enhance our understanding of loess records to a better perception of the impact of Last Glacial climate changes on European paleoenvironments.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Resveratrol inhibits lipogenesis of 3T3-L1 and SGBS cells by inhibition of insulin signaling and mitochondrial mass increase.

Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Interactions of Cu(B) with Carbon Monoxide in Cytochrome c Oxidase: Origin of the Anomalous Correlation between the Fe-CO and C-O Stretching Frequencies.

In heme-copper oxidases, the correlation curve between the iron-CO and C-O stretching vibrational modes (ν(Fe-CO) and ν(C-O), respectively) is anomalous as compared to the correlation in other heme proteins. To extend the correlation curve, the resonance Raman (RR) and infrared (IR) spectra of the CO adducts of cytochrome ba3 (ba3) from Thermus thermophilus were measured. The RR spectrum has two strong ν(Fe-CO) lines (508 and 515 cm(-1)) and a very weak line at 526 cm(-1), and the IR spectrum has three ν(C-O) lines (1966, 1973, and 1981 cm(-1)), indicating the presence of multiple conformers. Employing photodissociation methods, the ν(Fe-CO) RR and ν(C-O) IR lines were assigned to each conformer, enabling the establishment of a reliable inverse correlation curve for the ν(Fe-CO) versus the ν(C-O) stretching frequencies. To determine the molecular basis of the correlation, a series of DFT calculations on 6-coordinate porphyrin-CO compounds and a model of the binuclear center of the heme-copper oxidases were carried out. The calculations demonstrated that the copper unit model caused significant mixing among porphyrin-CO molecular orbitals (MOs) that contribute to the Fe-C and C-O bonding interactions, and also indicated the presence of mixing between the d(z)(2) orbital of the copper and MOs that are responsible for the ν(Fe-CO) vs ν(C-O) inverse correlation. Together, the spectroscopic and DFT results clarify the origin of the anomaly of ν(Fe-CO) and ν(C-O) frequencies in the heme-copper oxidases, a long-standing issue.

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae.

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQP(X)) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQP(X) and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the K(C)-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.