Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Phototriggered Apoptotic Cell Death (PTA) Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin-3.

Apoptosis is a type of programmed cell death that commonly occurs in multicellular organisms including humans and that is essential to eliminate unnecessary cells to keep organisms healthy. Indeed, inappropriate apoptosis leads to various diseases such as cancer and autoimmune disease. Here, we developed an optical method to regulate apoptotic cell death by controlling the intracellular pH with outward or inward proton pump rhodopsins, Archaerhodopsin-3 (AR3) or Rubricoccus marinas xenorhodopsin (RmXeR), respectively. The alkalization-induced shrinking of human HeLa cells cultured at pH 9.0 was significantly accelerated or decelerated by light-activated AR3 or RmXeR, respectively, implying the contribution of intracellular alkalization to the cell death. The light-activated AR3 induced cell shrinking at a physiologically neutral pH 7.4 and biochemical analysis revealed that the intracellular alkalization caused by AR3 triggered the mitochondrial apoptotic signaling pathway, which resulted in cell death accompanied by morphological changes. Phototriggered apoptosis (PTA) was also observed for other human cell lines, SH-SY5Y and A549 cells, implying its general applicability. We then used the PTA method with the nematode Caenorhabditis elegans as a model for living animals. Irradiation of transgenic worms expressing AR3 in chemosensing amphid sensory neurons significantly decreased their chemotaxis responses, which suggests that AR3 induced the cell death of amphid sensory neurons and the depression of chemotaxis responses. Thus, the PTA method has a high applicability both in vivo and in vitro, which suggests its potential as an optogenetic tool to selectively eliminate target cells with a high spatiotemporal resolution.

The three-spin intermediate at the O-O cleavage and proton-pumping junction in heme-Cu oxidases.

Understanding the mechanistic coupling of molecular oxygen reduction and proton pumping for adenosine triphosphate synthesis during cellular respiration is the primary goal of research on heme-copper oxidases­the terminal complex in the membrane-bound electron transport chain. Cleavage of the oxygen-oxygen bond by the heme-copper oxidases forms the key intermediate PM, which initiates proton pumping. This intermediate is now experimentally defined by variable-temperature, variable-field magnetic circular dichroism spectroscopy on a previously unobserved excited state feature associated with its heme iron(IV)-oxo center. These data provide evidence that the iron(IV)-oxo in PM is magnetically coupled to both a copper(II) and a cross-linked tyrosyl radical in the active site. These results provide new insight into the oxygen-oxygen bond cleavage and proton-pumping mechanisms of heme-copper oxidases.

Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life.

Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S0)-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

Allosteric Communication with the Retinal Chromophore upon Ion Binding in a Light-Driven Sodium Ion-Pumping Rhodopsin.

Krokinobacter rhodopsin 2 (KR2) serves as a light-driven sodium ion pump in the presence of sodium ion and works as a proton pump in the presence of larger monovalent cations such as potassium ion, rubidium ion, and cesium ion. Recent crystallographic studies revealed that KR2 forms a pentamer and possesses an ion binding site at the subunit interface. It is assumed that sodium ion bound at this binding site is not transported but contributes to the thermal stability. Because KR2 can convert its function in response to coexisting cation species, this ion binding site is likely to be involved in ion transport selectively. However, how sodium ion binding affects the structure of the retinal chromophore, which plays a crucial role in ion transport, remains poorly understood. Here, we observed the structure of the retinal chromophore under a wide range of cation concentrations using visible absorption and resonance Raman spectroscopy. We discovered that the hydrogen bond formed between the Schiff base of the retinal chromophore and its counterion, Asp116, is weakened upon binding of sodium ion. This allosteric communication between the Schiff base and the ion binding site at the subunit interface likely increases the apparent efficiency of sodium ion transport. In addition, this study demonstrates the significance of sodium ion binding: even though sodium ion is not transported, binding regulates the structure around the Schiff base and stabilizes the oligomeric structure.

Controlled micelle conjugation via charged peptide amphiphiles.

We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose-synthesized, peptide amphiphiles, C10 -(Asp)5 and C10 -(Lys)5 . Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C10 -(Asp)5 are conjugated (a) via a water-soluble, penta-Lys mediator or (b) to micelles containing the C10 -(Lys)5 peptide amphiphile. Both routes lead to phase separation in the form of oil-rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light-driven proton pump King Sejong 1-2 (KS1-2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.

Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane.

The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate.

We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time. We found that conditions that promoted direct interactions between the two proteins in the membrane (higher fraction DOPC lipids or labeling by hydrophobic molecules) correlated with an increased activity. These data indicate that the ATP-synthesis rate increases with decreasing distance between cytochrome bo 3 and the ATP-synthase, and involves proton transfer along the membrane surface. The maximum distance for lateral proton transfer along the surface was found to be ~80 nm.

The CarO rhodopsin of the fungus Fusarium fujikuroi is a light-driven proton pump that retards spore germination.

Rhodopsins are membrane-embedded photoreceptors found in all major taxonomic kingdoms using retinal as their chromophore. They play well-known functions in different biological systems, but their roles in fungi remain unknown. The filamentous fungus Fusarium fujikuroi contains two putative rhodopsins, CarO and OpsA. The gene carO is light-regulated, and the predicted polypeptide contains all conserved residues required for proton pumping. We aimed to elucidate the expression and cellular location of the fungal rhodopsin CarO, its presumed proton-pumping activity and the possible effect of such function on F. fujikuroi growth. In electrophysiology experiments we confirmed that CarO is a green-light driven proton pump. Visualization of fluorescent CarO-YFP expressed in F. fujikuroi under control of its native promoter revealed higher accumulation in spores (conidia) produced by light-exposed mycelia. Germination analyses of conidia from carO(-) mutant and carO(+) control strains showed a faster development of light-exposed carO(-) germlings. In conclusion, CarO is an active proton pump, abundant in light-formed conidia, whose activity slows down early hyphal development under light. Interestingly, CarO-related rhodopsins are typically found in plant-associated fungi, where green light dominates the phyllosphere. Our data provide the first reliable clue on a possible biological role of a fungal rhodopsin.

Structure of archaerhodopsin-2 at 1.8†Šresolution.

Archaerhodopsin-2 (aR2), the sole protein found in the claret membrane of Halorubrum sp. Aus-2, functions as a light-driven proton pump. In this study, structural analysis of aR2 was performed using a novel three-dimensional crystal prepared by the successive fusion of claret membranes. The crystal is made up of stacked membranes, in each of which aR2 trimers are arranged on a hexagonal lattice. This lattice structure resembles that found in the purple membrane of H. salinarum, except that lipid molecules trapped within the trimeric structure are not distributed with perfect threefold symmetry. Nonetheless, diffraction data at 1.8 Šresolution provide accurate structural information about functionally important residues. It is shown that two glutamates in the proton-release channel form a paired structure that is maintained by a low-barrier hydrogen bond. Although the structure of the proton-release pathway is highly conserved among proton-pumping archaeal rhodopsins, aR2 possesses the following peculiar structural features: (i) the motional freedom of the tryptophan residue that makes contact with the C13 methyl group of retinal is restricted, affecting the formation/decay kinetics of the L state, and (ii) the N-terminal polypeptide folds into an Ω-loop, which may play a role in organizing the higher-order structure.

Linking chemical electron-proton transfer to proton pumping in cytochrome c oxidase: broken-symmetry DFT exploration of intermediates along the catalytic reaction pathway of the iron-copper dinuclear complex.

After a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome c oxidase, we present the results of our earlier and recent density functional theory calculations for the dinuclear Fe-a3-CuB reaction center in this enzyme. A specific catalytic reaction wheel diagram is constructed from the calculations, based on the structures and relative energies of the intermediate states of the reaction cycle. A larger family of tautomers/protonation states is generated compared to our earlier work, and a new lowest-energy pathway is proposed. The entire reaction cycle is calculated for the new smaller model (about 185-190 atoms), and two selected arcs of the wheel are chosen for calculations using a larger model (about 205 atoms). We compare the structural and redox energetics and protonation calculations with available experimental data. The reaction cycle map that we have built is positioned for further improvement and testing against experiment.

Mechanisms of generation of local ΔpH in mitochondria and bacteria.

The concepts of global and local coupling between proton generators, the enzymes of the respiratory chain, and the consumer, the ATP synthase, coexist in the theory of oxidative phosphorylation. Global coupling is trivial proton transport via the aqueous medium, whereas local coupling implies that the protons pumped are consumed before they escape to the bulk phase. In this work, the conditions for the occurrence of local coupling are explored. It is supposed that the membrane retains protons near its surface and that the proton current generated by the proton pumps rapidly decreases with increasing proton motive force (pmf). It is shown that the competition between the processes of proton translocation across the membrane and their dissipation from the surface to the bulk can result in transient generation of a local ΔpH in reply to a sharp change in pmf; the appearance of local ΔpH, in turn, leads to rapid recovery of the pmf, and hence, it provides for stabilization of the potential at the membrane. Two mechanisms of such kind are discussed: 1) pH changes in the surface area due to proton pumping develop faster than those due to proton escape to the bulk; 2) the former does not take place, but the protons leaving the surface do not equilibrate with the bulk immediately; rather, they give rise to a non-equilibrium concentration near the surface and, as a result, to a back proton flow to the surface. The first mechanism is more efficient, but it does not occur in mitochondria and neutrophilic bacteria, whereas the second can produce ΔpH on the order of unity. In the absence of proton retardation at the surface, local ΔpH does not arise, whereas the formation of global ΔpH is possible only at buffer concentration of less than 10 mM. The role of the mechanisms proposed in transitions between States 3 and 4 of the respiratory chain is discussed. The main conclusion is that surface protons, under conditions where they play a role, support stabilization of the membrane pmf and rapid communication between proton generators and consumers, while their contribution to the energetics is not significant.

Route, mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps.

A single Na(+)/K(+)-ATPase pumps three Na(+) outwards and two K(+) inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na(+) than K(+) generates outward current across the cell membrane. Less well understood is the ability of Na(+)/K(+) pumps to generate an inward current of protons. Originally noted in pumps deprived of external K(+) and Na(+) ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K(+) and Na(+) concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na(+) release from phosphorylated Na(+)/K(+) pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na(+)/K(+) pumps that enables proton import is not required for completion of the 3 Na(+)/2 K(+) transport cycle. However, the back-step occurs readily during Na(+)/K(+) transport when external K(+) ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na(+)-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na(+) and K(+) ions that passes through binding site II. The inferred occurrence of Na(+)/K(+) exchange and H(+) import during the same conformational cycle of a single molecule identifies the Na(+)/K(+) pump as a hybrid transporter. Whether Na(+)/K(+) pump-mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.

Two different conformations in hepatitis C virus p7 protein account for proton transport and dye release.

The p7 protein from the hepatitis C virus (HCV) is a 63 amino acid long polypeptide that is essential for replication, and is involved in protein trafficking and proton transport. Therefore, p7 is a possible target for antivirals. The consensus model for the channel formed by p7 protein is a hexameric or heptameric oligomer of α-helical hairpin monomers, each having two transmembrane domains, TM1 and TM2, where the N-terminal TM1 would face the lumen of this channel. A reported high-throughput functional assay to search for p7 channel inhibitors is based on carboxyfluorescein (CF) release from liposomes after p7 addition. However, the rationale for the dual ability of p7 to serve as an ion or proton channel in the infected cell, and to permeabilize membranes to large molecules like CF is not clear. We have recreated both activities in vitro, examining the conformation present in these assays using infrared spectroscopy. Our results indicate that an α-helical form of p7, which can transport protons, is not able to elicit CF release. In contrast, membrane permeabilization to CF is observed when p7 contains a high percentage of ß-structure, or when using a C-terminal fragment of p7, encompassing TM2. We propose that the reported inhibitory effect of some small compounds, e.g., rimantadine, on both CF release and proton transport can be explained via binding to the membrane-inserted C-terminal half of p7, increasing its rigidity, in a similar way to the influenza A M2-rimantadine interaction.

Accessory subunits of mitochondrial complex I.

Mitochondrial complex I has a molecular mass of almost 1 MDa and comprises more than 40 polypeptides. Fourteen central subunits harbour the bioenergetic core functions. We are only beginning to understand the significance of the numerous accessory subunits. The present review addresses the role of accessory subunits for assembly, stability and regulation of complex I and for cellular functions not directly associated with redox-linked proton translocation.

Cation transport by the respiratory NADH:quinone oxidoreductase (complex I): facts and hypotheses.

The respiratory complex I (electrogenic NADH:quinone oxidoreductase) has been considered to act exclusively as a H+ pump. This was questioned when the search for the NADH-driven respiratory Na+ pump in Klebsiella pneumoniae initiated by Peter Dimroth led to the discovery of a Na+-translocating complex in this enterobacterium. The 3D structures of complex I from different organisms support the idea that the mechanism of cation transport by complex I involves conformational changes of the membrane-bound NuoL, NuoM and NuoN subunits. In vitro methods to follow Na+ transport were compared with in vivo approaches to test whether complex I, or its individual NuoL, NuoM or NuoN subunits, extrude Na+ from the cytoplasm to the periplasm of bacterial host cells. The truncated NuoL subunit of the Escherichia coli complex I which comprises amino acids 1-369 exhibits Na+ transport activity in vitro. This observation, together with an analysis of putative cation channels in NuoL, suggests that there exists in NuoL at least one continuous pathway for cations lined by amino acid residues from transmembrane segments 3, 4, 5, 7 and 8. Finally, we discuss recent studies on Na+ transport by mitochondrial complex I with respect to its putative role in the cycling of Na+ ions across the inner mitochondrial membrane.

Functional effects of mutations in cytochrome c oxidase related to prostate cancer.

A number of missense mutations in subunit I of cytochrome c oxidase (CytcO) have previously been linked to prostate cancer (Petros et al., 2005). To investigate the effects of these mutations at the molecular level, in the present study we prepared four different structural variants of the bacterial Rhodobacter sphaeroides CytcO (cytochrome aa(3)), each carrying one amino-acid residue replacement corresponding to the following substitutions identified in the above-mentioned study: Asn11Ser, Ala122Thr, Ala341Ser and Val380Ile (residues Asn25, Ser168, Ala384 and Val423 in the R. sphaeroides oxidase). This bacterial CytcO displays essentially the same structural and functional characteristics as those of the mitochondrial counterpart. We investigated the overall activity, proton pumping and internal electron- and proton-transfer reactions in the structural variants. The results show that the turnover activities of the mutant CytcOs were reduced by at most a factor of two. All variants pumped protons, but in Ser168Thr, Ala384Ser and Val423Ile we observed slight internal proton leaks. In all structural variants the internal electron equilibrium was slightly shifted away from the catalytic site at high pH (10), resulting in a slower observed ferryl to oxidized transition. Even though the effects of the mutations were relatively modest, the results suggest that they destabilize the proton-gating machinery. Such effects could be manifested in the presence of a transmembrane electrochemical gradient resulting in less efficient energy conservation. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

The identity of the transient proton loading site of the proton-pumping mechanism of cytochrome c oxidase.

Cellular respiration is driven by cytochrome c oxidase (CcO), which reduces oxygen to water and couples the released energy to proton pumping across the mitochondrial or bacterial membrane. Proton pumping in CcO involves proton transfer from the negatively charged side of the membrane to a transient proton-loading or pump site (PLS), before it is ejected to the opposite side. Although many details of the reaction mechanism are known, the exact location of the PLS has remained elusive. We report here results from combined classical molecular dynamics simulations and continuum electrostatic calculations, which show that the hydrogen-bonded system around the A-propionate of heme a3 dissociates reversibly upon reduction of heme a. The dissociation increases the pK(a) value of the propionate to a value above ~9, making it accessible for redox-state dependent protonation. The redox state of heme a is of key importance in controlling proton leaks by polarizing the PLS both statically and dynamically. These findings suggest that the propionate region of heme a3 fulfills the criteria of the pump site in the proton translocation mechanism of CcO.