Abstract
The cytochrome (cyt) bc1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme’s substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone – a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.
Highlights
The simulations supported the X-ray crystallographic results by showing that cardiolipin (CL) has a conserved binding position close to the Qi-site (Fig. 1C)[8,9]
The simulations indicate that the binding of half-protonated semiquinone (SQ) would acquire more coordinated binding pose than the deprotonated quinone (Q) at the Qi-site, because the Lys[251] side chain participates in the neutral SQ binding (Fig. 2A,B)
The substrate binding and H-bonding coordination could benefit from acquiring the protons sequentially shortly after each electron transfer
Summary
The simulations supported the X-ray crystallographic results by showing that cardiolipin (CL) has a conserved binding position close to the Qi-site (Fig. 1C)[8,9]. There exist plenty of X-ray crystal structures showing the bound substrate at the Qi-site (Figs 1D and S1; Table S1; see Supplementary Information (SI)), but the exact reduction steps are unknown due to lack of structural data on explicit protons (Fig. 1B) To address this issue, the bacterial cyt bc[1] complex was studied afresh using explicitly set up MD simulations (Table 1). Because only the anionic SQ has been detected using frozen electron paramagnetic resonance experiments at the Qi-site[12,13,14,15,16], the substrate has been presumed to acquire both of the electrons (dianionic state) before accepting the two protons concomitantly[17] While this scenario is possible, the other option is that the proton transfers are tightly coupled to separate e− transfers (Fig. 1B) as recently suggested by quantum mechanics calculations[16]. The accumulated data was used to formulate a novel Q reduction model in which the e−/H+ transfers are tightly coupled and sequential by nature as opposed to the previously suggested concomitant model[17]
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