Presented by Andrew Le

Xanthorhodopsin is a light-driven proton pump that associates with the vitamin retinol and salinixanthin, a carotenoid pigment Retinal: Vitamin A Sight Light-> metabolic energy Energy ACCEPTOR Carotenoid: organic pigment Antioxidants (conversion retinal) protect chlorophyll from photodamage. absorb light photosynthesis. Energy DONOR

Light energy-> pump protons across membrane-> electrochemical gradient -> generate chemical energy Simple: single protein with chromophore

Homologous to bacteriorhodopsin and proteorhodopsin (7 transmembrane helices) Difference through proton transfer through reaction cycle (mainly proton release) Contains histidine-aspartate to regulate pKa of primary proton acceptor X-ray diffraction revealed geometry of carotenoid and retinal explaining why the efficiency of the excited-state energy transfer is as high as 45%, and the 46° angle between them suggests that the chromophore location is a compromise between optimal light capture and excited- state energy transfer

Bacterial retinal-based proton pump, but also contains carotenoid Similarity to eubacterial photocycle Structure difference from archaeal proton pumps Small sequence homology to proteorhodopsins (22%) (Gloebacter violaceous (50%) Pyrocystis lunula)

Sequence alignment of green light-absorbing proteorhodopsin (PR), xanthorhodopsin (XR), and bacteriorhodopsin (BR) Not many similarities found (due to XR only possessing carotenoid binding)

Crystallized from bicelles with a type I arrangement of stacked bilayers Resolution Completeness Rwork/Rfree

The carotenoid is tightly bound on the transmembrane surface of Xanthorhodopsin, with inclination of 54° to membrane normal. Axis between chromophore axes is 46°. Axis between planes of Pi system 68°. Keto-Ring bind in pocket between E and F

Immobilized and formed by residues at the extracellular ends of helices E and F and by the β-ionone ring of the retinal. Retinal -ionone ring serves as part of the carotenoid binding site

The keto-ring of the carotenoid is rotated 82° out of plane of the salinixanthin-conjugated system and is in van der Waals distance of the retinal β–ionone and the phenolic side chain of Tyr C=O is not hydrogen bounded.

Energy Transfer is optimal when parralel and absorption of light optimal 90° (46° compromise between energy absorption and transfer) Conformational dependence of the carotenoid on the retinal and high efficiency (Close Contact > 5 Å) Well-resolved vibronic bands of the carotenoid, the lack of a red- shift of the absorption bands upon binding, and the strong CD bands in the visible region. (C=O)

Longer A and G helixes (4 and 9 residues) Different Tilt and rotation (mainly in A) In bacteriorhodopsin, the interhelical B–C antiparallel B-sheet interacts with the D–E loop. Xanthorhodopsin it reorients dramatically to interact with the Arg-8 peptide C=O near the N terminus, where it forms a mini 3-stranded B-sheet.

30Å displacement produces a large cleft that exposes functional residues and Schiff Base (usually buried) In bacteriorhodopsin, this region is occupied by the protein and includes the proton release group composed of Glu-194, Glu-204, and 3 ordered water molecules. Absent in Xanthorhodopsin-> unique proton release. NH1 and NH2 of Arg-93 are both hydrogen-bonded to the peptide carbonyl of Gln-229 instead of water molecules.

Wat-402 receives a hydrogen bond from the protonated retinal Schiff base and donates hydrogen bonds to the 2 anionic residues Asp-85 and Asp-212 (hydrogen-bonded aqueous network) Rearrangement of hydrogen bonds -> proton release

Arrangement is conserved in xanthorhodopsin. However, the carboxylate of the homolog of Asp- 85, Asp-96, is severely rotated. Hydrogen-bonded aqueous network replaced by hydrogenbonded residues that are resistant to rearrangement. (Arg-93 stabilized) No Proton Release at the time the Schiff base becomes deprotonated. Reverse Cycle 1) proton uptake by Glu-107 after it has reprotonated the retinal Schiff base 2) release to the extracellular surface is delayed until the final photocycle step. Light is used to release the proton rather than accept it

One of the characteristics of eubacterial proton pumps is that the pK a of the primary proton acceptor is near 7 (2.5 in bacteriorhodopsin) High pKa-> functions only at alkaline pH Histidine is highly conserved in proteorhodopsins-> aspartate-histidine complex general characteristic of eubacterial pumps Protonation states of the aspartate– histidine counterion complex. Crystallization at Ph 5.6 (lower) observed transition between the protonated and deprotonated forms of the Schiff base counterion-> neutral/zwitterionic counterion structure.