Pathways of proton release in the bacteriorhodopsin photocycle

Biochemistry. 1992 Sep 15;31(36):8535-43. doi: 10.1021/bi00151a022.

Abstract

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriorhodopsins / metabolism*
  • Bacteriorhodopsins / radiation effects*
  • Biological Transport
  • Halobacterium salinarum / chemistry
  • Hydrogen-Ion Concentration
  • Ion Pumps*
  • Kinetics
  • Models, Chemical
  • Photic Stimulation
  • Protons
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / radiation effects
  • Spectrophotometry

Substances

  • Ion Pumps
  • Protons
  • Recombinant Proteins
  • Bacteriorhodopsins