The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: Selective reactivity of different photointermediates (membrane protein/proton pump/M intermediate/water accessibility/time-resolved spectroscopy)

The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydrox- ylamine is enhanced by >2 orders of magnitude under illumi- nation. We have used this reaction as a probe for changes in Schiff base reactivity during the photocycle of wild-type bR and mutants defective in proton transport. We report here that under illumination at pH 6, the D85N mutant has a 20-fold lower rate and the D212N mutant has a >4-fold higher rate for the light-dependent reaction with hydroxylamine compared with wild-type bR. In contrast, the reactivities of wild-type bR and the D96N and T46V mutants are similar. It has been previously shown that the D96N and T46V replacements have no significant effect on the kinetics of "M" formation but have dramatic effects on rate of the decay of M. We therefore conclude that the hydroxylamine reaction occurs before for- mation of the M intermediate. Most likely it occurs at the "L" stage of the cycle and reflects increased water accessibility to the Schiff base due to a light-driven change in protein confor- G ~~~~~~~~~~~~~F T AV AR T W~~~~~~ I IL