1.) Studies with mammalian cytochrome aa3 (cox): In our previous report (1996) (published this year--Biochemistry (1997) 36:2439-2449), we described that our SVD-analyzed multichannel study of the kinetics of electron transfer events in the oxidation of fully reduced cox by O2 revealed steps with time constants (tau) of 0.01, 0.09, 1.1, and 30 ms and that a branched rather than the traditional linear sequence model of electron transport was correct. Another laboratory published that a linear sequence could explain their acquired data. We analyzed the published data from this laboratory and found that there was an insufficiency of time-based data (32 time-points compared to our 927) and that the curve- fitting algorithms were incorrectly applied (required weighting of the data was not done). Therefore, the evidence for a linear sequence was inadequate. The procedures and results of these analyses were written and submitted for publication (J. Biochem. Biophys. Meths.). 2.) Studies with bacteriorhodopsin (BR): We have identified by infrared (IR) studies, the principal structural damage to BR caused by brief exposure to Triton, which disrupts normal photocycle behavior, and is repaired by reconstitution with native lipids in high salt. It is a decrease in alpha-helical conformational flexibility as measured by a vibrational mode at approximately 1660 cm-1. Our past studies showed that there are two different photocycle pathways, one in which a kinetically fast M-intermediate decays to the O intermediate (Mf-->O) and one in which a slower M decays directly to BR (Ms-->BR). The former pathway predominates in low actinic light and the latter in high light. We have explored the question of whether this control is based on a higher efficiency for energy transduction under low light (using Mf) conditions than high intensity light (using Ms). We have looked both at the relative H+-pumping and delta psi 1-generating abilities of Mf and Ms. For the pumping efficiency, we have employed the dye pyranine which instantly responds to pH changes in combination with our ultrarapid spectrometer and SVD-based deconvolution procedures. For the delta psi 1 studies, we have set up an electrode-based system that can record the build-up and dissipation of voltages across a membrane with mus time resolution. Our results so far indicate that both Mf and Ms pump protons with the same efficiency, but that the delta psi 1-forming capability of Mf is superior to that of Ms. In the course of the pyranine studies, we have discovered a new photocycle intermediate with a time constant (tau) of approximately 600 mus. This tau coincides with the release of H+ into the medium and appears to represent a backwards turn in the photocycle in which the ground state BR forms the penultimate photocycle intermediate O in addition to the first forward intermediate J.
| Hendler, R W; Drachev, L A; Bose, S et al. (2000) On the kinetics of voltage formation in purple membranes of Halobacterium salinarium. Eur J Biochem 267:5879-90 |
| Joshi, M K; Bose, S; Hendler, R W (1999) Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium. Biochemistry 38:8786-93 |
