Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.
By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [zeta-(15)N]lys-, [12-(13)C]retinal-, and [14,15-(13)C]retinal-labeled D85N samples indicate a deprotonated, 13-cis,15-anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13-cis,15-syn, and all-trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a "bent" binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all-trans,15-anti configuration to the bent 13-cis,15-anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M(n) intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M(o) state. Thus, chromophore strain is released in the M(o)-->M(n) transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C [double bond] N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C [double bond] N syn species.
Hatcher
Mary E
ME
Department of Chemistry, Brandeis University, Waltham Massachusetts 02454, USA.
Hu
Jingui G
JG
Belenky
Marina
M
Verdegem
Peter
P
Lugtenburg
Johan
J
Griffin
Robert G
RG
Herzfeld
Judith
J
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009154:Mutation; D011522:Protons; D012176:Retinoids / Q000737:chemistry; D013696:Temperature
D011522:Protons; D012176:Retinoids; D001436:Bacteriorhodopsins; D002244:Carbon; D008239:Lysine