PROTON TRANSPORT BY A BACTERIORHODOPSIN MUTANT ASPARTIC ACID-85-]ASPARAGINE, INITIATED IN THE UNPROTONATED SCHIFF-BASE STATE

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-8 5 replaced by asparagine, is in a yellow form (lambda(max) approximate to 405 nm) with a deprotonated Schiff base, This state resembles the M intermediate of the wild-type photocycle, We used time-resolved meth ods to show that this yellow form of D85N, which has an initially unpr otonated Schiff base and which lacks the proton acceptor Asp-85, trans ports protons in the same direction as wild type when excited by 400-n m flashes, Photoexcitation leads in several milliseconds to the format ion of blue (630 nn) and purple (580 nm) intermediates with a protonat ed Schiff base, which decay in tens of seconds to the initial state (4 00 nm), Experiments with pH indicator dyes show that at pH 7, 8, and 9 , proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of prot on movement is from the cytoplasmic to the extracellular side with maj or components on the millisecond and second time scales. The slowest e lectrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state, Transport thus occurs in two steps, In the first step (millisec onds), the Schiff base is protonated by proton uptake from the cytopla smic side, thereby forming the blue state, From the pH dependence of t he amplitudes of the electrical and photocycle signals, we conclude th at this reaction proceeds in a similar way as in wild type-i.e., via t he internal proton donor Asp-96. In the second step (seconds) the Schi ff base deprotonates, releasing the proton to the extracellular side.