Crystal structure of the O intermediate of the Leu93→Ala mutant of bacteriorhodopsin

Abstract

The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λ(max) (540 nm) of the mutant. A long-lived substate of O with a red-shifted λ(max) (~565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (O(slow)) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O(slow), however, the main chain of Lys216 is less distorted and retinal takes on the 13-cis/15-syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK(a) value of which is suggested to be much lower in O(slow) than in M.

Existence of two O-like intermediates in the photocycle of <i>Acetabularia</i> rhodopsin II, a light-driven proton pump from a marine alga

“…Under neutral or alkaline conditions, N-decay is slow and the O 1 -to-O 2 conversion rate is presumably faster, which results in the lack of detection of O 1 . Allthough O-intermediate in the crystal structure of L93A BR takes 13- cis RET, 15- syn SBH + in agreement with the expected configuration of O 1 in the present paper [17], the precise RET configuration of O 1 of the wild-type BR should be determined in further study. In addition, we should explore its existence for other microbial rhodopsins.…”

“…In contrast, Subramaniam and coworkers reported the long life-time of O with 13- cis RET in L93A BR mutant which induces the slow reisomerization of RET due to abolishment of van der Waals interaction between the 13-methyl of RET and the terminal methyl groups of L93 BR [16]. In addition, Zhang et al solved its X-ray crystal structure and concluded that this long-lived O took 13- cis RET, 15- syn SBH + configuration [17]. These findings demonstrate at least the existence of a 13- cis O in L93A BR .…”

Photocycle of Sensory Rhodopsin II from Halobacterium salinarum (HsSRII): Mutation of D103 Accelerates M Decay and Changes the Decay Pathway of a 13‐cis O‐like Species

“…A similar photoproduct has been observed in the L93A mutant of BR . A crystallographic study showed that the O‐like intermediate has a 13‐ cis retinal chromophore instead of the all‐ trans one. Recently, Tamogami et al .…”

Unraveling the mechanism of proton translocation in the extracellular half‐channel of bacteriorhodopsin

“…A series of X‐ray crystal structures have been solved for intermediates trapped by mutation or freezing, which provide a structural basis for studying the proton‐pumping mechanism of bacteriorhodopsin. The structures show small conformational changes associated with the key residues in the CC and the EC .…”