Boris A. Feniouk

Chromatophore vesicles of Rhodobacter capsulatus contain on average one F(O)F(1)-ATP synthase each.

ATP synthase is a unique rotary machine that uses the transmembrane electrochemical potential difference of proton (Delta(H(+))) to synthesize ATP from ADP and inorganic phosphate. Charge translocation by the enzyme can be most conveniently followed in chromatophores of phototrophic bacteria (vesicles derived from invaginations of the cytoplasmic membrane). Excitation of chromatophores by a short flash of light generates a step of the proton-motive force, and the charge transfer, which is coupled to ATP synthesis, can be spectrophotometrically monitored by electrochromic absorption transients of intrinsic carotenoids in the coupling membrane. We assessed the average number of functional enzyme molecules per chromatophore vesicle. Kinetic analysis of the electrochromic transients plus/minus specific ATP synthase inhibitors (efrapeptin and venturicidin) showed that the extent of the enzyme-related proton transfer dropped as a function of the inhibitor concentration, whereas the time constant of the proton transfer changed only marginally. Statistical analysis of the kinetic data revealed that the average number of proton-conducting F(O)F(1)-molecules per chromatophore was approximately one. Thereby chromatophores of Rhodobacter capsulatus provide a system where the coupling of proton transfer to ATP synthesis can be studied in a single enzyme/single vesicle mode.

The cytochrome bc1 complex of Rhodobacter capsulatus: ubiquinol oxidation in a dimeric Q-cycle?

We studied the cytochrome bc 1 complex (hereafter bc) by flash excitation of Rhodobacter capsulatus chromatophores. The reduction of the high‐potential heme b h of cytochrome b (at 561 nm) and of cytochromes c (at 552 nm) and the electrochromic absorption transients (at 524 nm) were monitored after the first and second flashes of light, respectively. We kept the ubiquinone pool oxidized in the dark and concerned for the ubiquinol formation in the photosynthetic reaction center only after the second flash. Surprisingly, the first flash caused the oxidation of about one ubiquinol per bc dimer. Based on these and other data we propose a dimeric Q‐cycle where the energetically unfavorable oxidation of the first ubiquinol molecule by one of the bc monomers is driven by the energetically favorable oxidation of the second ubiquinol by the other bc monomer resulting in a pairwise oxidation of ubiquinol molecules by the dimeric bc in the dark. The residual unpaired ubiquinol supposedly remains on the enzyme and is then oxidized after the first flash.

Regulation of the F0F1‐ATP synthase: The conformation of subunit ε might be determined by directionality of subunit γ rotation

We propose that the directionality of the coiled‐coil subunit γ rotation determines whether subunit ε is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit ε. This conformation might serve as a safety lock, stabilizing the ADP‐inhibited state upon de‐energization and preventing spontaneous re‐activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit ε into a consistent picture.

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from about half of chromatophores

F(0)F(1)-ATP synthase (H(+)-ATP synthase, F(0)F(1)) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (P(i)). Structurally the enzyme consists of a membrane-embedded proton-translocating F(0) portion and a protruding hydrophilic F(1) part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F(0)F(1) (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F(1) (efrapeptin) and of F(0) (venturicidin), we decomposed the kinetics of the total proton flow through F(0)F(1) into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F(0). Taking the coupled proton flow, we calculated the H(+)/ATP ratio; it was found to be 3.3+/-0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1+/-0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F(0)F(1) dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and P(i) led to an increase in the fraction of chromatophores with at least one de-coupled F(0)(F(1)). The disclosed fraction of chromatophores that lacked proton-conducting F(0)(F(1)) (approx. 40% of the total amount) remained constant upon these treatments.

ATP‐synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP‐synthase (measured by electrochromic carotenoid bandshift and by pH‐indicators) and ATP release (measured by luminescence of luciferin‐luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP‐synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

Cellular and molecular mechanisms of action of mitochondria-targeted antioxidants

Reactive oxygen species generated in mitochondria are an important factor contributing to mitochondrial and cellular dysfunction underlying many degenerative diseases, chronic pathologies and aging. The idea of delivering antioxidant molecules to mitochondria in vivo to treat these diseases and slow aging intensively developed in the last 20 years. Derivatives of quinones covalently conjugated to a lipophilic cation (e.g., MitoQ and SkQ) were the most extensively studied mitochondria-targeted antioxidants. These compounds have now been used in a wide range of in vitro and in vivo studies, as well as in clinical trials in humans. Here, we review recent progress in this field with a special attention on molecular mechanisms of rechargeable mitochondria-targeted antioxidants. A simple hypothesis that aging results from gradual accumulation of occasional damage inflicted by ROS to DNA, proteins and lipids is apparently insufficient. More and more pieces of evidence indicate that the damage in question is programmed. Moreover, the imbalance in ROS-dependent regulatory mechanisms and compromised ROS signaling are underlying many pathologies and aging. Chain reactions of cardiolipin peroxidation initiated by mitochondrial ROS seem to play a key role in these degenerative processes. Such reactions are specifically abolished by mitochondriatargeted antioxidants.

Advanced glycation of cellular proteins as a possible basic component of the "master biological clock".

During the last decade, evidence has been accumulating supporting the hypothesis that aging is genetically programmed and, therefore, precisely timed. This hypothesis poses a question: what is the mechanism of the biological clock that controls aging? Measuring the level of the advanced glycation end products (AGE) is one of the possible principles underlying the functioning of the biological clock. Protein glycation is an irreversible, non-enzymatic, and relatively slow process. Moreover, many types of cells have receptors that can measure AGE level. We propose the existence of a protein that has a lifespan comparable to that of the whole organism. Interaction of the advanced glycation end product generated from this protein with a specific AGE receptor might initiate apoptosis in a vitally important non-regenerating tissue that produces a primary juvenile hormone. This could result in the age-dependent decrease in the level of this hormone leading to aging of the organism.

Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit

The C-terminal two α-helices of the ϵ-subunit of thermophilic Bacillus FoF1-ATP synthase (TFoF1) adopt two conformations: an extended long arm (“up-state”) and a retracted hairpin (“down-state”). As ATP becomes poor, ϵ changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TFoF1 expressed in Escherichia coli, we compared TFoF1 with up- and down-state ϵ in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TFoF1 with the up-state ϵ was achieved by inclusion of hexokinase in the assay and TFoF1 with the down-state ϵ was represented by ϵΔc-TFoF1, in which ϵ lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TFoF1 with the down-state ϵ synthesizes GTP at the same rate of ATP, whereas TFoF1 with the up-state ϵ synthesizes GTP at a half-rate. Though rates are slow, TFoF1 with the down-state ϵ even catalyzes UTP and CTP synthesis. Authentic TFoF1 from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of ϵΔc-TFoF1 suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ϵ-subunit modulates substrate specificity of TFoF1.

Thymic Involution in Ontogenesis: Role in Aging Program.

In most mammals, involution of the thymus occurs with aging. In this issue of Biochemistry (Moscow) devoted to phenoptosis, A. V. Khalyavkin considered involution of a thymus as an example of the program of development and further–of proliferation control and prevention of tumor growth. However, in animals devoid of a thymus (e.g. naked mice), stimulation of carcinogenesis, but not its prevention was observed. In this report, we focus on the involution of the thymus as a manifestation of the aging program (slow phenoptosis). We also consider methods of reversal/arrest of this program at different levels of organization of life (cell, tissue, and organism) including surgical manipulations, hormonal effects, genetic techniques, as well as the use of conventional and mitochondria-targeted antioxidants. We conclude that programmed aging (at least on the model of age-dependent thymic atrophy) can be inhibited.

Aging in birds

Rodents are the most commonly used model organisms in studies of aging in vertebrates. However, there are species that may suit this role much better. Most birds (Aves), having higher rate of metabolism, live two-to-three times longer than mammals of the same size. This mini-review briefly covers several evolutionary, ecological, and physiological aspects that may contribute to the phenomenon of birds’ longevity. The role of different molecular mechanisms known to take part in the process of aging according to various existing theories, e.g. telomere shortening, protection against reactive oxygen species, and formation of advanced glycation end-products is discussed. We also address some features of birds’ aging that make this group unique and perspective model organisms in longevity studies.