Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion.
Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NO(2)(-) and OCN(-) anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.
Mevorat-Kaplan
Keren
K
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Brumfeld
Vlad
V
Engelhard
Martin
M
Sheves
Mordechai
M
D000085:Acetates / Q000494:pharmacology; D000838:Anions / Q000494:pharmacology*; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry*; D003485:Cyanates / Q000494:pharmacology; D005561:Formates / Q000494:pharmacology*; D025602:Halorhodopsins / Q000737:chemistry / Q000187:drug effects*; D006863:Hydrogen-Ion Concentration; D019633:Natronobacterium / Q000737:chemistry; D009566:Nitrates / Q000494:pharmacology; D010777:Photochemistry; D010782:Photolysis; D013997:Time Factors
D000085:Acetates; D000838:Anions; D002712:Chlorides; D003485:Cyanates; D005561:Formates; D025602:Halorhodopsins; D009566:Nitrates