Qo site of the bc1 complex; unlocking the gate of the electron transport chain
The bc1 complex of the electron transport chain is critical to production of ATP through the proton gradient driven by quinone oxidation; a ubiquitous mechanism for energy conversion throughout the biosphere. In addition to being a central source of energy transduction, its bypass reactions can also lead to production toxic ROS formation from semiquinone intermediates. Its internal proton/electron shuttling and gating mechanisms have been a source of controversy and debate for decades. Structural and kinetic analysis of the bc1 complex suggests several proton-coupled electron transfer processes in which gating mechanisms favor rapid forward flux while minimizing deleterious by-pass reactions. Most important are those involved in the bifurcated reaction at the Qo site. The first electron transfer normally determines the overall rate. However, control of the fate of the intermediate semiquinone involves the second step. Interplay between charged intermediates arising from the separation of proton and electron transfer pathways, and involving conserved glutamate-295 (E295), allow coulombic control, while rotation of the E295 sidechain opens a volume into which the semiquinone can diffuse towards the electron acceptor, heme bL in the low potential chain, so as to facilitate its oxidation. When E295 is mutated, these processes are so strongly perturbed that the second electron transfer becomes the rate-limiting step, as demonstrated by measurement of flash-activated kinetics of the bc1 in chromatophores from wild type and E295 mutant strains of Rhodobacter sphaeroides, which show strong inhibi-tion of QH2 oxidation, and a dramatic difference in pH-dependence of activity. Attention has more recently been focused on the intermediate semiquinone, detected in the ~10-50 ms range using rapid-mix freeze-quench approaches, and shown to accumulate to low-occupancy under conditions in which its oxidation is inhibited. In our ~50ms freeze quenched studies, we showed that accumulation of SQo in wild-type occurs only after heme bL is reduced, but that in E295W, accumulation to even higher levels occurs while the heme remains oxidized. This shows that re-duction by SQo is severely inhibited, and allows an estimate of the inhibited rate-constant. Since in this strain, the SQo was constrained to the distal site in which it was generated, the occupancy levels likely reflect an impediment to the diffusional step that accelerates the rate constant by shortening the distance for electron transfer. This inhibition strongly supports a mechanism in which normal forward flux requires migration of SQo within the Qo site (2). Until recently, study of freeze-quenched radical intermediates on the sub-millisecond time scale has not been possible. Development of microfluidic ultra-fast freeze quenching devices over the last 10 years has allowed for collection at time points as low as ~50µs. However, the expertise, resources, and time required to fabricate these microfluidic devices, coupled with their high fragility once made, have made this technique relatively inaccessible. Here we report the development of an ultra-fast freeze quenching microfluidic apparatus, using a simple lithograph-ic construction. These microfluidic devices can be made in batches as single-use disposable components, and require little expertise, greatly increasing the accessibility of this useful tech-nology. We have used this approach in the first experiments to monitor the formation of a SQo intermediate, and establish kinetic parameters. The formation of SQo was monitored in bc1 com-plex from both WT and E295Q strains under different initial oxidation/reduction states by ob-serving the paramagnetic species by EPR. Spectral analysis using CW and pulsed EPR shows a SQo species not previously characterized, and has allowed resolution of some interesting struc-tural details. These suggest a species with spin coupling both to neighboring paramagnetic states, and local nuclear magnets. Kinetic measurements show that SQo is formed at a rate and occu-pancy consistent with a kinetic model developed to explore the diffusional hypothesis above. More detailed analysis using ESEEM suggests a strong coupling with the electron spin of the reduced 2Fe2S cluster of the iron-sulfur protein, likely involving the pyrrole nitrogen of His 152, and possibly longer-range interaction with the oxidized b-type hemes. However specific assign-ment of bonding/quantum coupling parameters will require more detailed spectroscopy, and quantum simulations in future collaborative efforts.
Q Cycle, Semiquinone, Biophysics, Structural, Electron Paramagnetic Resonance (EPR), Kinetics
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign