Enzymatic Activities of Isolated Cytochrome bc1-like Complexes Containing Fused Cytochrome b Subunits with Asymmetrically Inactivated Segments of Electron Transfer Chains

Homodimeric structure of cytochrome bc_1, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc1-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc_1 with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc_1 with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives

Magnetization dynamics and coherent spin manipulation of a propeller Gd(III) complex with the smallest helicene ligand

A homoleptic gadolinium(III) complex with the smallest helicene-type ligand, 1,10-phenanthroline-N,N'-dioxide (phendo) [Gd(phendo)(4)](NO3)(3)center dot xMeOH (phendo = 1,10-phenanthroline-N,N'-dioxide, MeOH = methanol), shows slow relaxation of the magnetization characteristic for Single Ion Magnets (SIM), despite negligible magnetic anisotropy, confirmed by ab initio calculations. Solid state dilution magnetic and EPR studies reveal that the magnetization dynamics of the [Gd(phendo)(4)](3+) cation is controlled mainly by a Raman process. Pulsed EPR experiments demonstrate long phase memory times (up to 2.7 mu s at 5 K), enabling the detection of Rabi oscillations at 20 K, which confirms coherent control of its spin state.</p

Cryo-EM structure of the spinach cytochrome b6 f complex at 3.6 Å resolution.

The cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1-3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 Å resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis

Molecular organization of cytochrome c_{2} near the binding domain of cytochrome bc_{1} studied by electron spin-lattice relaxation enhancement

[Image: see text] Measurements of specific interactions between proteins are challenging. In redox systems, interactions involve surfaces near the attachment sites of cofactors engaged in interprotein electron transfer (ET). Here we analyzed binding of cytochrome c(2) to cytochrome bc(1) by measuring paramagnetic relaxation enhancement (PRE) of spin label (SL) attached to cytochrome c(2). PRE was exclusively induced by the iron atom of heme c(1) of cytochrome bc(1), which guaranteed that only the configurations with SL to heme c(1) distances up to ∼30 Å were detected. Changes in PRE were used to qualitatively and quantitatively characterize the binding. Our data suggest that at low ionic strength and under an excess of cytochrome c(2) over cytochrome bc(1), several cytochrome c(2) molecules gather near the binding domain forming a “cloud” of molecules. When the cytochrome bc(1) concentration increases, the cloud disperses to populate additional available binding domains. An increase in ionic strength weakens the attractive forces and the average distance between cytochrome c(2) and cytochrome bc(1) increases. The spatial arrangement of the protein complex at various ionic strengths is different. Above 150 mM NaCl the lifetime of the complexes becomes so short that they are undetectable. All together the results indicate that cytochrome c(2) molecules, over the range of salt concentration encompassing physiological ionic strength, do not form stable, long-lived complexes but rather constantly collide with the surface of cytochrome bc(1) and ET takes place coincidentally with one of these collisions

The Interplay Between Respiratory Supercomplexes and ROS in Aging

Significance: The molecular mechanism of aging is still vigorously debated, although a general consensus exists that mitochondria are significantly involved in this process. However, the previously postulated role of mitochondrial-derived reactive oxygen species (ROS) as the damaging agents inducing functional loss in aging has fallen out of favor in the recent past. In this review, we critically examine the role of ROS in aging in the light of recent advances on the relationship between mitochondrial structure and function. Recent Advances: The functional mitochondrial respiratory chain is now recognized as a reflection of the dynamic association of respiratory complexes in the form of supercomplexes (SCs). Besides providing kinetic advantage (channeling), SCs control ROS generation by the respiratory chain, thus providing a means to regulate ROS levels in the cell. Depending on their concentration, these ROS are either physiological signals essential for the life of the cell or toxic species that damage cell structure and functions. Critical Issues: We propose that under physiological conditions the dynamic nature of SCs reversibly controls the generation of ROS as signals involved in mitochondrial- nuclear communication. During aging, there is a progressive loss of control of ROS generation so that their production is irreversibly enhanced, inducing a vicious circle in which signaling is altered and structural damage takes place. Future Directions: A better understanding on the forces affecting SC association would allow the manipulation of ROS generation, directing these species to their physiological signaling role

Specific LED-based red light photo-stimulation procedures improve overall sperm function and reproductive performance of boar ejaculates

The present study evaluated the effects of exposing liquid-stored boar semen to different red light LED regimens on sperm quality and reproductive performance. Of all of the tested photo-stimulation procedures, the best pattern consisted of 10 min light, 10 min rest and 10 min of further light (10-10-10 pattern). This pattern induced an intense and transient increase in the majority of motility parameters, without modifying sperm viability and acrosome integrity. While incubating non-photo-stimulated sperm at 37 °C for 90 min decreased all sperm quality parameters, this reduction was prevented when the previously-described light procedure was applied. This effect was concomitant with an increase in the percentage of sperm with high mitochondrial membrane potential. When sperm were subjected to ‘in vitro’ capacitation, photo-stimulation also increased the percentage of sperm with capacitation-like changes in membrane structure. On the other hand, treating commercial semen doses intended for artificial insemination with the 10-10-10 photo-stimulation pattern significantly increased farrowing rates and the number of both total and live-born piglets for parturition. Therefore, our results indicate that a precise photo-stimulation procedure is able to increase the fertilising ability of boar sperm via a mechanism that could be related to mitochondrial functionThis research received support from Spanish Ministry of Economy and Competitiveness (Grant AGL2013-47798-P