Bacteriorhodopsin (BR) is a 26000 D, energy-transducing proton pump in the purple membrane of H. Salinarium 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 much simpler molecule than Cox both in size and complexity, it offers an ideal prototype for probing the mechanism of how proton pumps work. A key to understanding the process is to define the precise kinetic mechanism of molecular transformations that result in the electrogenic transfer of a proton across the membrane. Although, it is known that BR upon energization by photon-absorption passes through a number of intermediate states labeled sequentially as K, L, M1, M2, N, and O, before returning to the ground state, BR, the precise sequence and manner of linkage has not been clearly defined. The predominant view in the field is that of a single sequential chain of reversible reactions as BR <--> K <--> L <--> M1 <--> M2 <--> N <--> BR. Based on this model, it is postulated that the M1 to M2 transformation acts as a gate in which the orientation of the proton-binding group changes from the extracellular to cytoplasmic surface to ensure unidirectionality in the transport process. Our laboratory has published evidence that the kinetic mechanism at pH 7 and 20 degrees C involves a pair of unidirectional cycles with M1 and M2 in different cycles, rather than the reversible homogeneous cycle described above. During the year, we have completed our analyses of the kinetics of the BR-photoycle under nine different conditions of temperature and pH. The results show that under all conditions, groups of from 2 to 4 parallel, essentially unidirectional, cycles function rather than the single, reversible, homogeneous (RHM) model as commonly believed. Under some of the tested conditions, isolated reversible reactions occur for the M to N transformation. The appropriate kinetic model for each condition is expressed in the form of a discrete set of ordinary differential equations (ODE). Using the ODE, along with the fitted exponential first order kinetic constants from the raw data, allows one to derive the kinetic profile for each intermediate in the photocycle. With a matrix representation of these profiles (Y), we can extract from the raw data, the isolated absolute spectrum for each intermediate. Given a set of raw experimental data in a matrix where columns are the spectra and rows are the time points, pure, isolated spectra are retrieved. This methodology can be combined with other time-resolved approaches to yield important new information about the whole proton-pumping mechanism. There are two aspects of this potential that we plan to pursue in the coming year. Time-resolved Fourier Transform Infrared (FTIR) reports on both protein conformational changes and specific sites of proton-binding. In parallel experiments, both the optical and FTIR spectra will be collected and presented for analysis in a single matrix. The retrieval of isolated optical spectra will also yield isolated FTIR spectra. In this way, it will be possible to observe conformational and proton-binding as the photocycle progresses. Using the same approach, we plan to combine the optical spectra with time-resolved X-ray crystallography to retrieve isolated X-ray diffraction patterns for each intermediate. This, in principle, should provide structural information at the atomic level for the protein as it moves the proton across the membrane. Finally, we are able to measure the proton current and its kinetics to help identify which of the transformation steps in the photocycle are most coupled to the development of membrane potential across the membrane. During the course of this work, we observed that the distribution of parallel cycles on a 2-dimensional grid of temperature vs. pH resembles a phase diagram. Our previous work has shown that particular lipids in the membrane react with particular amino acid residues of BR to influence the conformation of the protein, and the pathways and kinetics of the photocycle. Infrared spectroscopy (IR) reveals information on the conformation of lipids and proteins and interactions between them. It is planned to use IR to study these aspects of lipids and proteins at different pH?s and temperatures to see if phase changes in lipids are associated with the differences in photocycles found as ambient conditions are changed.
