Bacteriorhodopsin (BR) is a 26000 D, energy-transducing proton pump in the purple membrane of H. Salinarum that is capable of forming the same kind of protonmotive force (PMF) as mammalian cytochrome oxidase (Cox). PMF is the driving force for synthesis of ATP using the F1Fo ATPase. Because BR is a much simpler molecule than Cox both in size and complexity, it offers an ideal prototype for probing the mechanism of how proton pumps work. Work from our laboratory has shown that the BR photocycle at pH 7 and 20?X consists of two parallel cycles. One goes through the intermediates BR??Lf??Mf??N??O??BR and the other BR??Ls??Ms??BR. The former cycle contains a form of M that decays faster than the M in the latter cycle and is called the M-fast cycle. The latter is the M-slow cycle. In understanding the mechanism of proton-pumping, we would like to know, which cycle is the more efficient pump and which steps in the cycle form the most membrane potential. The kinetics of the two photocycles show different saturation behaviors in their responses to increasing strengths of actinic laser flashes. Starting at the lowest levels of laser strength, the M-fast cycle rises faster than the M-slow cycle, but reaches saturation with increasing laser strength sooner than and at a lower level than the M-slow cycle. As a consequence, a plot of Mf/Mtot, where Mtot includes both Mf and Ms, starts high and fall with laser strength, whereas the fraction Ms/Mtot starts low and rises with laser strength. The intermediate, O, in the M-fast cycle when plotted as the fraction O/Mtot shows the same response to increasing actinic light as does the fraction Mf/Mtot, as is to be expected since they are both in the same cycle.? ? It is possible to measure directly the laser-induced proton current across the membrane. This is done by orienting the membrane fragments in a DC electric field and then trapping them in an acrylamide gel. Two platinized electrodes with a suitable amplifier then can measure the time-resolved proton current (I). The kinetics of I match those measured optically. From I we can derive values for voltage (V) formed at each stage of proton movement. Plots of the fractions I/Mtot and V/Mtot as functions of laser strength reveal that at least 97% of proton movement is associated with the M-slow cycle. Thus, the M-slow cycle is the primary energy transducer and the final step of Ms??BR appears to generate most of the membrane potential.? ? For at least 30 years, a group of researchers maintained that whenever kinetics are studied following a laser flash, the kinetic data can be biased by a process of photoselection. All light, when reflected or passed through lenses becomes somewhat polarized. Polarized laser light will predominantly activate molecules whose absorption dipole is in the same orientation as the laser light. Similarly, the monitoring light, if polarized, will be absorbed predominantly by molecules with their dipoles in the same orientation. If, during the time of measurement, the activated molecules change their orientation because of diffusion or Brownian motion, a discrepancy in measurement will arise due to changes in orientation with respect to the monitoring light. There are equations which show that if the plane of the monitoring light is maintained at 54.7 degrees from that of the laser light, (i.e. the magic angle (MA)), all possible kinetic artifacts resulting from photoselection artifacts can be eliminated. We have always disagreed with the idea that artifacts of photoselection contribute to BR kinetics, and according to our measurements find that the use of MA, itself, introduces kinetic artifacts. This past year (2005), we published our findings that the BR photocycle under a variety of different pH!|s and temperatures were all composed of from 2 to 4 parallel cycles. Two of the principal proponents for the use of MA optics (L and N) challenged our findings because we did not use the MA technique. At the invitation of the Journal for Physical Chemistry B, we wrote an extensive reply to the comments made by these challengers. Both the comments of L and N and our reply were critically reviewed by four referees of the journal, who all recommended publication. Both articles were published in the journal as well as an extensive Supplementary Material paper of ours, which was published electronically. Our reply included four sections. First, we reviewed their basis for objecting. Next we reported that no published work has ever established that non-photocycle kinetic events corrupted the true kinetics of the photocycle. We then cited many publications which present positive evidence that non-photocycle events do not contribute to measured kinetics obtained without the use of MA optics. Finally, we presented evidence that the use of MA procedures does alter the true kinetics. In our Supplementary Material paper, we examined, in detail, the equations used to measure the positions of the chromophores during the photocycle cited by L and N and then used them to substantiate our position that no non-photocycle-related changes in chromophore orientation seems to occur. We also presented data and analyses which we performed to identify such non-photocycle events and the results which show their absence. We used the actual data of L and N to show that no evidence of their suggested artifactual chromophore reorientations occurred. We then used the same data to show that the MA data contained irregularities not in accord with predictions of the equations that were cited by L and N as a basis for their objections.
