Selective reaction of hydroxylamine with chromophore during the photocycleof pharaonis phoborhodopsin

Phoborhodopsin (pR; also called sensory rhodopsin II, sRII) is a receptor o f negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodop sin (ppR; also pharaonis sensory rhodopsin II, psRII) is a corresponding pr otein of Natronobacterium pharaonis. These receptors contain retinal as a c hromophore which binds to a lysine residue via Schiff base. This Schiff bas e can be cleaved with hydroxylamine to loose their color (bleaching). In da rk, the bleaching rate of ppR was very slow whereas illumination accelerate d considerably the bleaching rate. Addition of azide accelerated the decay of the M-intermediate while its formation (decay of the L-intermediate) is not affected. The bleaching rate of ppR under illumination was decreased by addition of azide. Essentially no reactivity with hydroxylamine under illu mination was observed in the case of D75N mutant which lacks the M-intermed iate in its photocycle. Moreover, we provided illumination by flashes to pp R in the presence of varying concentrations of azide to measure the bleachi ng rate per one flash. A good correlation was obtained between the rate and the mean residence time, MRT, which was calculated from flash photolysis d ata of the M-decay. These findings reveal that water-soluble hydroxylamine reacts selectively with the M-intermediate and its implication was discusse d. (C) 2001 Published by Elsevier Science B.V.