Stimulation of cytochrome c oxidase activity by detergents.

Cytochrome c oxidase (CytcO) is an integral membrane protein, which catalyzes four-electron reduction of oxygen linked to proton uptake and pumping. Amphipathic molecules bind in sites near the so-called K proton pathway of CytcO to reversibly modulate its activity. However, purification of CytcO for mechanistic studies typically involves the use of detergents, which may interfere with binding of these regulatory molecules. Here, we investigated the CytcO enzymatic activity as well as intramolecular electron transfer linked to proton transfer upon addition of different detergents to bovine heart mitoplasts. The CytcO activity increased upon addition of alkyl glucosides (DDM and DM) and the steroid analog GDN. The maximum stimulating effect was observed for DDM and DM, and the half-stimulating effect correlated with their CMC values. With GDN the stimulation effect was smaller and occurred at a concentration higher than CMC. A kinetic analysis suggests that the stimulation of activity is due to removal of a ligand bound near the K proton pathway, which indicates that in the native membrane this site is occupied to yield a lower than maximal possible CytcO activity. Possible functional consequences are discussed.

Overcoming Protein Orientation Mismatch Enables Efficient Nanoscale Light-Driven ATP Production.

Adenosine triphosphate (ATP)-producing modules energized by light-driven proton pumps are powerful tools for the bottom-up assembly of artificial cell-like systems. However, the maximum efficiency of such modules is prohibited by the random orientation of the proton pumps during the reconstitution process into lipid-surrounded nanocontainers. Here, we overcome this limitation using a versatile approach to uniformly orient the light-driven proton pump proteorhodopsin (pR) in liposomes. pR is post-translationally either covalently or noncovalently coupled to a membrane-impermeable protein domain guiding orientation during insertion into preformed liposomes. In the second scenario, we developed a novel bifunctional linker, trisNTA-SpyTag, that allows for the reversible connection of any SpyCatcher-containing protein and a HisTag-carrying protein. The desired protein orientations are verified by monitoring vectorial proton pumping and membrane potential generation. In conjunction with ATP synthase, highly efficient ATP production is energized by the inwardly pumping population. In comparison to other light-driven ATP-producing modules, the uniform orientation allows for maximal rates at economical protein concentrations. The presented technology is highly customizable and not limited to light-driven proton pumps but applicable to many membrane proteins and offers a general approach to overcome orientation mismatch during membrane reconstitution, requiring little to no genetic modification of the protein of interest.

Mitochondria at the Nanoscale: Physics Meets Biology-What Does It Mean for Medicine?

Mitochondria are commonly perceived as "cellular power plants". Intriguingly, power conversion is not their only function. In the first part of this paper, we review the role of mitochondria in the evolution of eukaryotic organisms and in the regulation of the human body, specifically focusing on cancer and autism in relation to mitochondrial dysfunction. In the second part, we overview our previous works, revealing the physical principles of operation for proton-pumping complexes in the inner mitochondrial membrane. Our proposed simple models reveal the physical mechanisms of energy exchange. They can be further expanded to answer open questions about mitochondrial functions and the medical treatment of diseases associated with mitochondrial disorders.

Declines in ice cover are accompanied by light limitation responses and community change in freshwater diatoms.

The rediscovery of diatom blooms embedded within and beneath the Lake Erie ice cover (2007-2012) ignited interest in psychrophilic adaptations and winter limnology. Subsequent studies determined the vital role ice plays in winter diatom ecophysiology as diatoms partition to the underside of ice, thereby fixing their location within the photic zone. Yet, climate change has led to widespread ice decline across the Great Lakes, with Lake Erie presenting a nearly "ice-free" state in several recent winters. It has been hypothesized that the resultant turbid, isothermal water column induces light limitation amongst winter diatoms and thus serves as a competitive disadvantage. To investigate this hypothesis, we conducted a physiochemical and metatranscriptomic survey that spanned spatial, temporal, and climatic gradients of the winter Lake Erie water column (2019-2020). Our results suggest that ice-free conditions decreased planktonic diatom bloom magnitude and altered diatom community composition. Diatoms increased their expression of various photosynthetic genes and iron transporters, which suggests that the diatoms are attempting to increase their quantity of photosystems and light-harvesting components (a well-defined indicator of light limitation). We identified two gene families which serve to increase diatom fitness in the turbid ice-free water column: proton-pumping rhodopsins (a potential second means of light-driven energy acquisition) and fasciclins (a means to "raft" together to increase buoyancy and co-locate to the surface to optimize light acquisition). With large-scale climatic changes already underway, our observations provide insight into how diatoms respond to the dynamic ice conditions of today and shed light on how they will fare in a climatically altered tomorrow.

Using cryo-EM to understand the assembly pathway of respiratory complex I.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

The mitochondrial respiratory chain.

We present a brief review of the mitochondrial respiratory chain with emphasis on complexes I, III and IV, which contribute to the generation of protonmotive force across the inner mitochondrial membrane, and drive the synthesis of ATP by the process called oxidative phosphorylation. The basic structural and functional details of these complexes are discussed. In addition, we briefly review the information on the so-called supercomplexes, aggregates of complexes I-IV, and summarize basic physiological aspects of cell respiration.

Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

F-type proton-pumping ATPase mediates acid tolerance in Streptococcus mutans.

AIMS: Streptococcus mutans is highly sensitive to inhibitors of proton-pumping F-type ATPase (F-ATPase) under acidic conditions. Herein, we investigated the role of S. mutans F-ATPase in acid tolerance using a bacterium expressing the F-ATPase ß subunit at lower levels than the wild-type strain. METHODS AND RESULTS: We generated a mutant S. mutans expressing the catalytic ß subunit of F-ATPase at lower levels than the wild-type bacterium. The mutant cells exhibited a significantly slower growth rate at pH 5.30, whereas the rate was essentially the same as that of wild-type cells at pH 7.40. In addition, the colony-forming ability of the mutant was decreased at pH <4.30 but not at pH 7.40. Thus, the growth rate and survival of S. mutans expressing low levels of the ß subunit were reduced under acidic conditions. CONCLUSIONS: Together with our previous observations, this study indicates that F-ATPase is involved in the acid tolerance mechanism of S. mutans by secreting protons from the cytoplasm.

From the 'black box' to 'domino effect' mechanism: what have we learned from the structures of respiratory complex I.

My group and myself have studied respiratory complex I for almost 30 years, starting in 1994 when it was known as a L-shaped giant 'black box' of bioenergetics. First breakthrough was the X-ray structure of the peripheral arm, followed by structures of the membrane arm and finally the entire complex from Thermus thermophilus. The developments in cryo-EM technology allowed us to solve the first complete structure of the twice larger, ∼1 MDa mammalian enzyme in 2016. However, the mechanism coupling, over large distances, the transfer of two electrons to pumping of four protons across the membrane remained an enigma. Recently we have solved high-resolution structures of mammalian and bacterial complex I under a range of redox conditions, including catalytic turnover. This allowed us to propose a robust and universal mechanism for complex I and related protein families. Redox reactions initially drive conformational changes around the quinone cavity and a long-distance transfer of substrate protons. These set up a stage for a series of electrostatically driven proton transfers along the membrane arm ('domino effect'), eventually resulting in proton expulsion from the distal antiporter-like subunit. The mechanism radically differs from previous suggestions, however, it naturally explains all the unusual structural features of complex I. In this review I discuss the state of knowledge on complex I, including the current most controversial issues.

The evolution of the human mitochondrial bc1 complex- adaptation for reduced rate of superoxide production?

The mitochondrial bc1 complex is a major source of mitochondrial superoxide. While bc1-generated superoxide plays a beneficial signaling role, excess production of superoxide lead to aging and degenerative diseases. The catalytic core of bc1 comprises three peptides -cytochrome b, Fe-S protein, and cytochrome c1. All three core peptides exhibit accelerated evolution in anthropoid primates. It has been suggested that the evolution of cytochrome b in anthropoids was driven by a pressure to reduce the production of superoxide. In humans, the bc1 core peptides exhibit anthropoid-specific substitutions that are clustered near functionally critical sites that may affect the production of superoxide. Here we compare the high-resolution structures of bovine, mouse, sheep and human bc1 to identify structural changes that are associated with human-specific substitutions. Several cytochrome b substitutions in humans alter its interactions with other subunits. Most significantly, there is a cluster of seven substitutions, in cytochrome b, the Fe-S protein, and cytochrome c1 that affect the interactions between these proteins at the tether arm of the Fe-S protein and may alter the rate of ubiquinone oxidation and the rate of superoxide production. Another cluster of substitutions near heme bH and the ubiquinone reduction site, Qi, may affect the rate of ubiquinone reduction and thus alter the rate of superoxide production. These results are compatible with the hypothesis that cytochrome b in humans (and other anthropoid primates) evolve to reduce the rate of production of superoxide thus enabling the exceptional longevity and exceptional cognitive ability of humans.

Single protonation of the reduced quinone in respiratory complex I drives four-proton pumping.

Complex I is a key proton-pumping enzyme in bacterial and mitochondrial respiratory electron transport chains. Using quantum chemistry and electrostatic calculations, we have examined the pKa of the reduced quinone QH-/QH2 in the catalytic cavity of complex I. We find that pKa (QH-/QH2) is very high, above 20. This means that the energy of a single protonation reaction of the doubly reduced quinone (i.e. the reduced semiquinone QH-) is sufficient to drive four protons across the membrane with a potential of 180 mV. Based on these calculations, we propose a possible scheme of redox-linked proton pumping by complex I. The model explains how the energy of the protonation reaction can be divided equally among four pumping units of the pump, and how a single proton can drive translocation of four additional protons in multiple pumping blocks.

On the role of ubiquinone in the proton translocation mechanism of respiratory complex I.

Complex I converts oxidoreduction energy into a proton electrochemical gradient across the inner mitochondrial or bacterial cell membrane. This gradient is the primary source of energy for aerobic synthesis of ATP. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) by ubiquinone (Q) yields NAD+ and ubiquinol (QH2 ), which is tightly coupled to translocation of four protons from the negatively to the positively charged side of the membrane. Electrons from NADH oxidation reach the iron-sulfur centre N2 positioned near the bottom of a tunnel that extends circa 30 Å from the membrane domain into the hydrophilic domain of the complex. The tunnel is occupied by ubiquinone, which can take a distal position near the N2 centre or proximal positions closer to the membrane. Here, we review important structural, kinetic and thermodynamic properties of ubiquinone that define its role in complex I function. We suggest that this function exceeds that of a mere substrate or electron acceptor and propose that ubiquinone may be the redox element of complex I coupling electron transfer to proton translocation.

Tunnel dynamics of quinone derivatives and its coupling to protein conformational rearrangements in respiratory complex I.

Respiratory complex I in mitochondria and bacteria catalyzes the transfer of electrons from NADH to quinone (Q). The free energy available from the reaction is used to pump protons and to establish a membrane proton electrochemical gradient, which drives ATP synthesis. Even though several high-resolution structures of complex I have been resolved, how Q reduction is linked with proton pumping, remains unknown. Here, microsecond long molecular dynamics (MD) simulations were performed on Yarrowia lipolytica complex I structures where Q molecules have been resolved in the ~30 Å long Q tunnel. MD simulations of several different redox/protonation states of Q reveal the coupling between the Q dynamics and the restructuring of conserved loops and ion pairs. Oxidized quinone stabilizes towards the N2 FeS cluster, a binding mode not previously described in Yarrowia lipolytica complex I structures. On the other hand, reduced (and protonated) species tend to diffuse towards the Q binding sites closer to the tunnel entrance. Mechanistic and physiological relevance of these results are discussed.

Reaction pathways, proton transfer, and proton pumping in ba3 class cytochrome c oxidase: perspectives from DFT quantum chemistry and molecular dynamics.

After drawing comparisons between the reaction pathways of cytochrome c oxidase (CcO, Complex 4) and the preceding complex cytochrome bc1 (Complex 3), both being proton pumping complexes along the electron transport chain, we provide an analysis of the reaction pathways in bacterial ba3 class CcO, comparing spectroscopic results and kinetics observations with results from DFT calculations. For an important arc of the catalytic cycle in CcO, we can trace the energy pathways for the chemical protons and show how these pathways drive proton pumping of the vectorial protons. We then explore the proton loading network above the Fe heme a3-CuB catalytic center, showing how protons are loaded in and then released by combining DFT-based reaction energies with molecular dynamics simulations over states of that cycle. We also propose some additional reaction pathways for the chemical and vector protons based on our recent work with spectroscopic support.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 µmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

Osteoclast rich osteopetrosis due to defects in the TCIRG1 gene.

Discovery that mutations in TCIRG1 (also known as Atp6i) gene are responsible for most instances of autosomal recessive osteopetrosis (ARO) heralded a new era for comprehension and treatment of this phenotypically heterogeneous rare bone disease. TCIRG1 encodes the a3 subunit, an essential isoform of the vacuolar ATPase proton pump involved in acidification of the osteoclast resorption lacuna and in secretory lysosome trafficking. TCIRG1 defects lead to inefficient bone resorption by nonfunctional osteoclasts seen in abundance on bone marrow biopsy, delineating this ARO as 'osteoclast-rich'. Presentation is usually in early childhood and features of extramedullary haematopoiesis (hepatosplenomegaly, anaemia, thrombocytopenia) due to bone marrow fibrosis, and cranial nerve impingement (blindness in particular). Impaired dietary calcium uptake due to high pH causes the co-occurrence of rickets, described as "osteopetrorickets". Osteoclast dysfunction leads to early death if untreated, and allogeneic haematopoietic stem cell transplantation is currently the treatment of choice. Studies of patients as well as of mouse models carrying spontaneous (the oc/oc mouse) or targeted disruption of Atp6i (TCIRG1) gene have been instrumental providing insight into disease pathogenesis and development of novel cellular therapies that exploit gene correction.

Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae.

Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the nad1, nad2, nad4, and nad5 genes, reside at the mitochondrial inner membrane and possibly function as proton (H+) and ion translocators. To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene (encoding enhanced green fluorescent protein, EGFP) were fused at the 5'-terminal and 3'-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.

Photoactivated Bacteriorhodopsin/SiNx Nanopore-Based Biological Nanofluidic Generator with Single-Protein Sensitivity.

Nanofluidics is an emerging hot field that explores the unusual behaviors of ions/molecules transporting through nanoscale channels, which possesses a broad application prospect. However, in situ probing bioactivity of functional proteins on a single-molecule level by a nanofluidic device has not been reported, and it is still a big challenge in the field. Herein, we reported a biological nanofluidic device with a single-protein sensitivity, based on natural proton-pumping protein, bacteriorhodopsin (bR), and a single SiNx nanopore. Nanofluidic single-molecule probing of bR proton-pumping activity and its light response were achieved under applied voltage of 0 V, by biologically self-powered steady-state ionic current nanopore sensing. Green-light irradiation of the device led to the monitoring of a steady-state proton current of ∼3.51 pA/per bR trimer, corresponding to charge density of 815 µC/cm2 generated by each bR monomer, which far exceeded the previously reported value of 1.4 µC/cm2. This finding and method would promote the development of artificial biological and hybrid nanofluidic devices in biosensing and energy conversion applications.

Complex Interplay of Heme-Copper Oxidases with Nitrite and Nitric Oxide.

Nitrite and nitric oxide (NO), two active and critical nitrogen oxides linking nitrate to dinitrogen gas in the broad nitrogen biogeochemical cycle, are capable of interacting with redox-sensitive proteins. The interactions of both with heme-copper oxidases (HCOs) serve as the foundation not only for the enzymatic interconversion of nitrogen oxides but also for the inhibitory activity. From extensive studies, we now know that NO interacts with HCOs in a rapid and reversible manner, either competing with oxygen or not. During interconversion, a partially reduced heme/copper center reduces the nitrite ion, producing NO with the heme serving as the reductant and the cupric ion providing a Lewis acid interaction with nitrite. The interaction may lead to the formation of either a relatively stable nitrosyl-derivative of the enzyme reduced or a more labile nitrite-derivative of the enzyme oxidized through two different pathways, resulting in enzyme inhibition. Although nitrite and NO show similar biochemical properties, a growing body of evidence suggests that they are largely treated as distinct molecules by bacterial cells. NO seemingly interacts with all hemoproteins indiscriminately, whereas nitrite shows high specificity to HCOs. Moreover, as biologically active molecules and signal molecules, nitrite and NO directly affect the activity of different enzymes and are perceived by completely different sensing systems, respectively, through which they are linked to different biological processes. Further attempts to reconcile this apparent contradiction could open up possible avenues for the application of these nitrogen oxides in a variety of fields, the pharmaceutical industry in particular.

Membrane-domain mutations in respiratory complex I impede catalysis but do not uncouple proton pumping from ubiquinone reduction.

Respiratory complex I [NADH:ubiquinone (UQ) oxidoreductase] captures the free energy released from NADH oxidation and UQ reduction to pump four protons across an energy-transducing membrane and power ATP synthesis. Mechanisms for long-range energy coupling in complex I have been proposed from structural data but not yet evaluated by robust biophysical and biochemical analyses. Here, we use the powerful bacterial model system Paracoccus denitrificans to investigate 14 mutations of key residues in the membrane-domain Nqo13/ND4 subunit, defining the rates and reversibility of catalysis and the number of protons pumped per NADH oxidized. We reveal new insights into the roles of highly conserved charged residues in lateral energy transduction, confirm the purely structural role of the Nqo12/ND5 transverse helix, and evaluate a proposed hydrated channel for proton uptake. Importantly, even when catalysis is compromised the enzyme remains strictly coupled (four protons are pumped per NADH oxidized), providing no evidence for escape cycles that circumvent blocked proton-pumping steps.