Photocycle and Abnormal Activity of the Dual Chromophore Proton Pump Archaerhodopsin-4 with and Without the Second Chromophore

Abstract

While several archaeal ion pumps have been revealed to contain two chromophores, retinal and carotenoid bacterioruberin, their photocycle, activity and mediation mechanism by the second chromophore bacterioruberin have not been well explored. In order to address these challenges, the recombinant archaerhodopsin-4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ light-induced transient absorption change spectroscopy, low temperature FTIR difference spectroscopy and 2D solid-state NMR, together with molecular dynamic simulation, were employed to study the photocycle, photo-intermediates and activity of aR4 with and without the second chromophore bacterioruberin. Our results revealed for the first time that the photocycle of wild-type aR4 only contains one M state but two N states, and unlike in bacteriorhodopsin the accessibility switch of the SB nitrogen in the photocycle of aR4 may occur in the N1 → N2 states. Perturbed L, M and N states were observed in the bacterioruberin-excluded aR4 indicating that a strong coupling may exist between bacterioruberin and aR4 photocycle. Furthermore, a constitutively activated aR4 was also observed in the bacterioruberin-excluded aR4 which can be characterized by the structural instability and conformational response to the deletion of the second chromophore. These new insights may be generalized to other receptors and proteins in which functions are perturbed by ligand binding.