THE NATURE OF THE MULTICOMPONENT M-INTERMEDIATE FORMATION IN THE BACTERIORHODOPSIN PHOTOCYCLE .1. ANALYSIS OF THE M-INTERMEDIATE FORMATION OF THE WILD-TYPE BACTERIORHODOPSIN

The kinetics of L-intermediate decay and M-intermediate formation in t he wild-type bacteriorhodopsin photocycle (pH 7.0, 20 degrees C) are w ell fitted by a sum of two components with tau(1) = 60 mu sec and tau( 2) = 250 mu sec. The fast M formation is associated with L-decay havin g a difference maximum at 550 nm, while the slow formation is associat ed with L-decay having a difference maximum at 530 nm, which points to the existence of two parallel pathways in M-intermediate formation. A n additional component (tau(3) = 1 msec; cf. 2.5 msec in glycerol) evo lves during M-formation in the presence of Lu3(+) or in 80% glycerol. The K-intermediate decay is significantly retarded in this case, while M-formation is accelerated in glycerol (the time constants are 7 and 65 mu sec, respectively). The L-intermediate is hardly detectable unde r these conditions. The 1 msec component is present in the photocycle as a lag phase in the relaxation of the photoresponse at 400 and 520 n m and as an additional component in the optical density increase at 33 5 nm in the presence 100 mM NaCl (pH 7.0). A model of M-formation for 3(+) or in 80% glycerol (2) is proposed: [GRAPHICS] The appearance of the 1 msec component in the M-formation is well explained by the quasi -stationary equilibrium L reversible arrow M in the presence of Lu3+ o r in glycerol. The appearance of the proton in the bulk water solution measured with the pH indicator pyranine in the absence of buffers mat ches the third phase of M-formation. In the presence of the buffer the rate of pyranine protonation coincides with the rate of M-formation. This suggests that the proton release from the protein into the membra ne-water interface is associated with the L --> M transition, while th at proton transfer into the bulk solution os associated with the M --> M step in the absence of buffers. The M --> M step is suggested to re flect a conformational rearrangement of the protein.