To understand how the protein achieves fluorescence, the isomerization mechanism of the HcRed chromophore is studied both under vacuum and in the solvated red fluorescent protein. Quantum mechanical (QM) and quantum mechanical/molecular mechanical (QM/MM) methods are applied both for the ground and the first excited state. The photoinduced processes in the chromophore mainly involve torsions around the imidazolinone-bridge bond (t) and the phenoxy-bridge bond (j). Under vacuum, the isomerization of the cis-trans chromophore essentially proceeds by t twisting, while the radiationless decay requires j torsion. By contrast, the isomerization of the cis-trans chromophore in HcRed occurs via simultaneous t and j twisting. The protein environment significantly reduces the barrier of this hula twist motion compared with vacuum. The excited-state isomerization barrier via the j rotation of the cis-coplanar conformer in HcRed is computed to be significantly higher than that of the trans-non-coplanar conformer. This is consistent with the experimental observation that the cis-coplanar-conformation of the chromophore is related to the fluorescent properties of HcRed, while the trans-non-planar conformation is weakly fluorescent or non-fluorescent. Our study shows how the protein modifies the isomerization mechanism, notably by interactions involving the nearby residue Ile197, which keeps the chromophore coplanar and blocks the twisting motion that leads to photoinduced radiationless decay.

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...