Marcin Sarewicz

An Electronic Bus Bar Lies in the Core of Cytochrome bc1

Heme Communication Revealed by Asymmetry An electronic bus bar is an electrical conductor that connects several circuits. Świerczek et al. (p. 451) now find that a similar strategy is used by the protein cytochrome bc1 that plays a central role in cellular respiration and photosynthesis. Protein engineering was used to break the symmetry of a cytochrome bc1 homodimer, which revealed that the dimer is bridged by electron transfer between two hemes. This allows electrons to move freely within and between dimers to distribute between four catalytic sites. Electrons move across the entire structure of a functional dimer of an enzyme central to cellular bioenergetics. The ubiquinol–cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are functionally important. This has resulted in a proliferation of contradictory models. Here, we duplicated and fused cytochrome b subunits, and then broke symmetry by introducing independent mutations into each monomer. Electrons moved freely within and between monomers, crossing an electron-transfer bridge between two hemes in the core of the dimer. This revealed an H-shaped electron-transfer system that distributes electrons between four quinone oxidation-reduction terminals at the corners of the dimer within the millisecond time scale of enzymatic turnover. Free and unregulated distribution of electrons acts like a molecular-scale bus bar, a design often exploited in electronics.

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1 Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQo) formed at the quinone oxidation/reduction Qo site (Qo) as a result of single-electron oxidation of quinol by the iron–sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme bL (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc1 that severely impeded the oxidation of FeS by cytochrome c1, changed density of FeS around Qo by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Qo and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQo that present a risk of generation of superoxide by cytochrome bc1. Isolation of this reaction sequence from multiplicity of possible reactions at Qo helps to better understand conditions under which complex III might contribute to ROS generation in vivo.

Movement of the Iron-Sulfur Head Domain of Cytochrome bc1 Transiently Opens the Catalytic Qo Site for Reaction with Oxygen

Cytochrome bc(1), a key enzyme of biological energy conversion, generates or uses a proton motive force through the Q cycle that operates within the two chains of cofactors that embed two catalytic quinone oxidation/reduction sites, the Q(o) site and the Q(i) site. The Q(o) site relies on the joint action of two cofactors, the iron-sulfur (FeS) cluster and heme b(L). Side reactions of the Q cycle involve a generation of superoxide which is commonly thought to be a product of an oxidation of a highly unstable semiquinone formed in the Q(o) site (SQ(o)), but the overall mechanism of superoxide generation remains poorly understood. Here, we use selectively modified chains of cytochrome bc(1) to clearly isolate states linked with superoxide production. We show that this reaction takes place under severely impeded electron flow that traps heme b(L) in the reduced state and reflects a probability with which a single electron on SQ(o) is capable of reducing oxygen. SQ(o) gains this capability only when the FeS head domain, as a part of a catalytic cycle, transiently leaves the Q(o) site to communicate with the outermost cofactor, cytochrome c(1). This increases the distance between the FeS cluster and the remaining portion of the Q(o) site, reducing the likelihood that the FeS cluster participates in an immediate removal of SQ(o). In other states, the presence of both the FeS cluster and heme b(L) in the Q(o) site increases the probability of completion of short-circuit reactions which retain single electrons within the enzyme instead of releasing them on oxygen. We propose that in this way, cytochrome bc(1) under conditions of impeded electron flow employs the leak-proof short-circuits to minimize the unwanted single-electron reduction of oxygen.

Atomistic simulations indicate cardiolipin to have an integral role in the structure of the cytochrome bc1 complex.

The reaction mechanism of the cytochrome (cyt) bc1 complex relies on proton and electron transfer to/from the substrate quinone/quinol, which in turn generate a proton gradient across the mitochondrial membrane. Cardiolipin (CL) have been suggested to play an important role in cyt bc1 function by both ensuring the structural integrity of the protein complex and also by taking part in the proton uptake. Yet, the atom-scale understanding of these highly charged four-tail lipids in the cyt bc1 function has remained quite unclear. We consider this issue through atomistic molecular dynamics simulations that are applied to the entire cyt bc1 dimer of the purple photosynthetic bacterium Rhodobacter capsulatus embedded in a lipid bilayer. We find CLs to spontaneously diffuse to the dimer interface to the immediate vicinity of the higher potential heme b groups of the complexs catalytic Qi-sites. This observation is in full agreement with crystallographic studies of the complex, and supports the view that CLs are key players in the proton uptake. The simulation results also allow us to present a refined picture for the dimer arrangement in the cyt bc1 complex, the novelty of our work being the description of the role of the surrounding lipid environment: in addition to the specific CL-protein interactions, we observe the protein domains on the positive side of the membrane to settle against the lipids. Altogether, the simulations discussed in this article provide novel views into the dynamics of cyt bc1 with lipids, complementing previous experimental findings.

Electronic Connection Between the Quinone and Cytochrome c Redox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria.

Key role of water in proton transfer at the Qo-site of the cytochrome bc1 complex predicted by atomistic molecular dynamics simulations

Cytochrome (cyt) bc1 complex, which is an integral part of the respiratory chain and related energy-conserving systems, has two quinone-binding cavities (Qo- and Qi-sites), where the substrate participates in electron and proton transfer. Due to its complexity, many of the mechanistic details of the cyt bc1 function have remained unclear especially regarding the substrate binding at the Qo-site. In this work we address this issue by performing extensive atomistic molecular dynamics simulations with the cyt bc1 complex of Rhodobacter capsulatus embedded in a lipid bilayer. Based on the simulations we are able to show the atom-level binding modes of two substrate forms: quinol (QH2) and quinone (Q). The QH2 binding at the Qo-site involves a coordinated water arrangement that produces an exceptionally close and stable interaction between the cyt b and iron sulfur protein subunits. In this arrangement water molecules are positioned suitably in relation to the hydroxyls of the QH2 ring to act as the primary acceptors of protons detaching from the oxidized substrate. In contrast, water does not have a similar role in the Q binding at the Qo-site. Moreover, the coordinated water molecule is also a prime candidate to act as a structural element, gating for short-circuit suppression at the Qo-site.

Demonstration of Short-lived Complexes of Cytochrome c with Cytochrome bc1 by EPR Spectroscopy IMPLICATIONS FOR THE MECHANISM OF INTERPROTEIN ELECTRON TRANSFER

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc1. To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc1, we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths. Our data show a successive decrease of the bound cytochrome c fraction as the ionic strength increases, with a limit of ∼120 mm NaCl above which essentially no bound cytochrome c can be detected by EPR. This limit does not apply to all of the interactions of cytochrome c with cytochrome bc1 because the cytochrome bc1 enzymatic activity remained high over a much wider range of ionic strengths. We concluded that EPR monitors just the tightly bound state of the association and that an averaged lifetime of this state decreases from over 100 μs at low ionic strength to less than 400 ns at an ionic strength above 120 mm. This suggests that at physiological ionic strength, the tightly bound complex on average lasts less than the time needed for a single electron exchange between hemes c and c1, indicating that productive electron transfer requires several collisions of the two molecules. This is consistent with an early idea of diffusion-coupled reactions that link the soluble electron carriers with the membranous complexes, which, we believe, provides a robust means of regulating electron flow through these complexes.

Magnetic Interactions Sense Changes in Distance between Heme bL and the Iron−Sulfur Cluster in Cytochrome bc1

During the operation of cytochrome bc1, a key enzyme of biological energy conversion, the iron−sulfur head domain of one of the subunits of the catalytic core undergoes a large-scale movement from the catalytic quinone oxidation Qo site to cytochrome c1. This changes a distance between the two iron−two sulfur (FeS) cluster and other cofactors of the redox chains. Although the role and the mechanism of this movement have been intensely studied, they both remain poorly understood, partly because the movement itself is not easily traceable experimentally. Here, we take advantage of magnetic interactions between the reduced FeS cluster and oxidized heme bL to use dipolar enhancement of phase relaxation of the FeS cluster as a spectroscopic parameter which with a unique clarity and specificity senses changes in the distance between those two cofactors. The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc1 were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc1. This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc1 and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations. To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place. The measured changes in the phase relaxation enhancement provide the first direct experimental description of changes in the strength of dipolar coupling between the FeS cluster and heme bL.

Triplet state of the semiquinone-Rieske cluster as an intermediate of electronic bifurcation catalyzed by cytochrome bc1.

Efficient energy conversion often requires stabilization of one-electron intermediates within catalytic sites of redox enzymes. While quinol oxidoreductases are known to stabilize semiquinones, one of the famous exceptions includes the quinol oxidation site of cytochrome bc1 (Qo), for which detection of any intermediate states is extremely difficult. Here we discover a semiquinone at the Qo site (SQo) that is coupled to the reduced Rieske cluster (FeS) via spin–spin exchange interaction. This interaction creates a new electron paramagnetic resonance (EPR) transitions with the most prominent g = 1.94 signal shifting to 1.96 with an increase in the EPR frequency from X- to Q-band. The estimated value of isotropic spin–spin exchange interaction (|J0| = 3500 MHz) indicates that at a lower magnetic field (typical of X-band) the SQo–FeS coupled centers can be described as a triplet state. Concomitantly with the appearance of the SQo–FeS triplet state, we detected a g = 2.0045 radical signal that corresponded to the population of unusually fast-relaxing SQo for which spin–spin exchange does not exist or is too small to be resolved. The g = 1.94 and g = 2.0045 signals reached up to 20% of cytochrome bc1 monomers under aerobic conditions, challenging the paradigm of the high reactivity of SQo toward molecular oxygen. Recognition of stable SQo reflected in g = 1.94 and g = 2.0045 signals offers a new perspective on understanding the mechanism of Qo site catalysis. The frequency-dependent EPR transitions of the SQo–FeS coupled system establish a new spectroscopic approach for the detection of SQo in mitochondria and other bioenergetic systems.

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 bc1, 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 bc1 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 bc1 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.