The Mechanism of Photo-energy Storage in the Halorhodopsin Chloride Pump

Abstract

The light-driven pump Halorhodopsin (hR) uses the energy stored in an initial meta-stable state (K), in which the bound retinal chromophore has been photoisomerized from all-trans to 13-cis, to drive the translocation of one chloride anion across the membrane. Thus far it was unclear whether retinal twisting or charge separation between the positive Schiff base of the retinal and the chloride anion is the primary mechanism of energy storage. Here, combined quantum mechanical/molecular mechanical (QM/MM) simulations show that the energy is predominantly stored by charge separation. However, a large variability in retinal twisting is observed, thus reconciling the contradictory hypotheses for storage. Surprisingly, the energy stored in the K-state amounts to only one-fifth of the photon energy. We explain why the protein cannot store more: even though this would accelerate chloride pumping, raising the K-state also reduces the relative energy barriers against unproductive decay, in particular via the premature cis to trans backisomerization. Indeed, the protein has maximized its storage so that the back-isomerization barriers are just high enough (>18 kcal/mol) to keep the decay rate (1/100 ms) slower than the remaining photocycle (1/20 ms). This need to stabilize the captured photon-energy until it can be used in subsequent steps is inherent to light-driven proteins.Light driven pumps such as halorhodopsin (hR) 2 stand apart from other energy-driven transporters. To them, the primary source of energy is not permanently available, unlike energy stored in the form of chemical bonds (for example, ATP in the case of the Na ϩ /K ϩ -ATPase (1)) or in electrochemical transmembrane gradients (like the Na ϩ /glucose symport transporter (2)). Thus, they must be able to first harvest the energy of the photon, before converting that energy into work. The harvesting of the fleeting photon necessitates converting the photon energy into a meta-stable form of molecular energy, and to prevent the dissipation of this molecular energy until it can be subsequently used to power the pumping mechanism. In other words, the protein must be able to simultaneously store and stabilize the energy. For the absorption of light, proteins employ a chromophoric ligand or group, which changes from one state to another upon photon absorption. This transition is unfavorable and slow in the electronic ground-state, but is favored when the chromophore has been excited by the photon. After the return of the system to the electronic ground-state (within picoseconds in the case of hR (3)), the chromophore relaxes either to the starting ground-state conformation (thus reducing the quantum yield) or to a meta-stable high-energy state (with a quantum yield of 34% in the case of hR (3)). The energy stored in this meta-stable form relative to the groundstate form is what powers the subsequent pumping steps (Fig. 1A). Understanding how this energy is stored thus constitutes an essential aspect of the mechanism and has been the focus of studies on visual rh...

Crystal Structures of an O-Like Blue Form and an Anion-Free Yellow Form of pharaonis Halorhodopsin

“…Since the water distribution around Glu234 is largely altered, it is likely that the pK a value of Glu234 is changed by the purple-to-blue transition. Diffraction data of the chloride-free blue or yellow form at various pH levels (pH [4][5][6][7][8][9][10] showed that the conformation of Glu234 remained unaltered in the investigated pH range. This observation suggests that Glu234 is deprotonated in the blue form and in the yellow form.…”

“…[1][2][3][4][5][6] HR is structurally similar to bacteriorhodopsin (BR), a light-driven proton pump found in Halobacterium salinarum. 7,8 Both HR and BR are made up of seven transmembrane helices (helix A through G) and the retinal chromophore that is bound via a protonated Schiff base to the ɛ-amino group of a lysine residue located in the middle of helix G. 9 In these retinylidene proteins, the photoisomerization of retinal around the C13 = C14 double bond triggers a series of conformational changes by which a proton or a chloride ion is transported.…”

Catalytic strategy used by the myosin motor to hydrolyze ATP

“…The method allows determination of the mechanism of complex reactions in proteins. CPR has been used successfully in conjunction with QM/MM energy functions, for example to study proton transfer along proton wires (52,53), the cleavage of P ̶ O bonds (9,31), or isomerization reactions (54)(55)(56). Unlike molecular dynamics simulations, the MEP shows only the motions that are essential for the reaction, but gives no information on the time needed to get from one movie frame to the next.…”

Electrostatic interactions and hydrogen bond dynamics in chloride pumping by halorhodopsin

“…In the all- trans resting state HR and the first two 13- cis states, K and L1, the chloride ion appears to remain largely in the same location as in the starting HR state [12-14]. Nevertheless, perturbed interactions of the Schiff base and R123 may weaken the chloride interactions at site-1, and set the stage for the chloride displacement from site-1 to the cytoplasmic side of the retinal in state L2 [15].…”