1.) Studies with mammalian cytochrome aa3 (cox): Using our unique multichannel spectrometer and SVD analysis, we published that the reduction of O2 by fully reduced cox follows a branched electron-transport pathway (Biochemistry (1997) 36:2439). In contrast, another laboratory published that the pathway was linear. We published two papers this year demonstrating that the mathematical modeling used in the other laboratory was incorrect (J. Biochem. Biophys. Meth. (1998) 36:157) and that the rate of O2-binding to cox is 5 times faster than previously thought (Biochem. Mol. Biol. Intern. (1998) 45:1031). 2.) Studies with bacteriorhodopsin (BR): (A) Attention was centered on studies with BR in the intact Halobacteria in relation to the more typical studies with isolated purple membrane (PM) fragments. We find that the photocycle in cells is much slower than in isolated membranes. This is due to a control exercised by the membrane potential (MP), which is built from an existing potassium gradient, cell respiration, and ATP-hydrolysis. The slow kinetics are traced to newer and slower species of photocycle intermediates. The kinetics in whole cells can be """"""""titrated"""""""" to become equal to those in membrane fragments by the energy-uncoupler CCCP or simply by lysing the cells. Agents which cause an increase in MP (i.e. DCCD) cause a further slowing of kinetics. Freshly-prepared cells which have the highest levels of potassium gradient, respiration activity, and ATP pools, show a partial reversal of the forward photocycle in the earliest phase (i.e. ~8 ms). (B) We discovered that 0.01% decane can transform the slow M phtotocycle intermediate to the fast M species in wildtype PM. Such a transformation has never been seen before. (C) We have established that normal photocycle kinetics and regulation by light energy depends on a direct interaction of a phosphatydyl glycerophosphate/squalene pair with a unique acidic aminoacid residue in BR (Biochemistry, in press). To identify the aminoacid, we have started to work with single-site mutants, selected on the basis of X-Ray diffraction data. We find, that a D38N mutant shows kinetics like those found in Triton-damaged wild type. Normal kinetics could be established in 0.01% decane. (D) A new collaboration was started with J. Heberle in Germany to use time-resolved FTIR to study the relation of protein conformational changes and ligand-binding to the kinetics of turnover and concomitant proton-binding and release. (E) A new collaboration was started with Ad Bax (NIDDK) to investigate oriented PM membranes by NMR to learn more about the structure of this important integral membrane protein under normal, Triton-damaged and reconstituted conditions. (F) A new collaboration was started with Ira Levin (NIDDK) to study the conformations of BR in model lipid membranes.
| 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 |
