Interrelations of M‐intermediates in bacteriorhodopsin photocycle

Abstract

The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by tile flash-photolysis technique. Tile M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, rt = 65 and r, = 250/~s) for the wild-type bR and thr~ exponents (r~ = 55 ,us, r,. = 220 as and r.a = 1 ms) for the D96N mutant of bR. A component with r = 1 ms was found to be prescott in the photoeycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu ~* ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the I-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the l-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp~-~Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identi~l for th~ wild-type bR and D96N mutant and matched the M->M' eonlbrmational transition, A model for M rise in the bR photocycle is proposed.

Chemical and functional studies on the importance of purple membrane lipids in bacteriorhodopsin photocycle behavior

“…It has been known for some time that there are (at least) two forms of the M intermediate in the bacteriorhodopsin (BR) photocycle, one (Mf) with faster kinetics than the other (Ms), and that the level of actinic light alters the fractions of these two species (see [2][3][4] and references therein). More recently, it has been shown that Mf decays to the ground state through the O intermediate whereas Ms does not [4,5].…”

Cooperative phenomena in the photocycle of D96N mutant bacteriorhodopsin

“…The mutant D96N gives this opportunity owing to an ability of a number of weak acids (azide, cyanate, nitrite, etc.) to serve as proton donors for the Shiffbase [10][11][12][13][14][15]. The proportional dependence of the rate of the M decay on the concentration of the protonated form of azide indicates that it is a second-order reaction.…”

Two bacteriorhodopsin M intermediates differing in accesibility of the Schiff base for azide

“…It is well known now that the M intermediate, in fact, comprises a set of different forms with similar absorption spectra [4][5][6][7][8][9]. There are many pieces of evidence for protein conformational changes accompanying the proton transport, especially the reprotonation of the Schiff base by the internal proton donor D96, i.e.…”

“…the decay of the M intermediate [9][10][11][12][13][14]. The mutation leading to the lack of proton donor (D96N) causes a dramatic retardation of the M decay [15,16], and this kinetic defect can be repaired by azide and other anions of small weak acids [7,[17][18][19][20][21][22]. It was initially supposed that the action of azide was associated with its ability to serve as the penetrating proton donor [7,[17][18][19][20][21], however, another model has recently been put forward [22] according to which the tightly bound anionic form of azide can catalyze proton transfer to the Schiff base.…”

“…The bR photocycle was monitored using a single-beam spectrophotometer [7,19,24]. Photoexcitation of bR was carried out with a YG-481 Quantel neodymium laser (~.…”