The Ability of Actinic Light to Modify the Bacteriorhodopsin Photocycle Revisited: Heterogeneity vs. Photocooperativity

A long standing observation, that the photocycle of bacteriorhodopsin is markedly altered by the strength of actinic light, has not been adequately explained (1). The initial and primary illustration of the phenomenon is that the fraction of M-fast (Mf) to total M (Mtot) starts high at low laser pulse strength and smoothly decreases with increasing strength (2). In addition, Mf decays through the N and O intermediates to BR,1 whereas M-slow (Ms) does not (3,4). For the 25 years since Ohno clearly demonstrated the ability of light to regulate the ratio of Mf/Ms, there have been two different possible explanations for the phenomenon. One, based on a homogeneous photocycle and photocooperativity, proposes that the fundamental target, a trimer of BR molecules, when hit by a single photon decays through the Mf route, whereas when two or three photons strike the target, decay includes the Ms route. The other explanation proposes that there are different ground states (BR) that proceed through different photocycles. Recently (4), one of us (R.W.H.) reviewed evidence published both prior to and since the earlier review on the question of a photocooperative-homogeneous vs heterogeneous parallel cycles. A group of publications were cited which could be explained by parallel photocycles but not by single homogeneous cycles. Evidence for the existence of different ground states was presented. Minimal criteria were specified that should be met before any proposed kinetic model be seriously considered. The model must provide a solution to the equation A = DY, where D is a matrix containing all absolute spectra for the intermediates and Y is a matrix containing time courses for each component in D. The time courses in Y are made up of a mixture of all of the exponentials involved in each discrete transition specified by the model. Rate constants for these exponentials must be provided, and when the model is expressed using a Jacobian matrix J, that J must yield eigenvalues which equal the observed kinetic macro constants obtained by fitting exponentials to the experimental data. When the matrix D is obtained from the raw data by the operation D = A/Y, where /Y is the pseudo inverse of Y, the derived absolute spectra must correspond to those found in other laboratories using independent procedures. Parallel cycle models were shown to meet all of these criteria under nine different combinations of temperature and pH. In contrast, not a single proposed photocooperative–homogeneous model has yet been shown to do the same. At neutral pH and T near 20 °C, two different cycles have been identified (3,4). BR→Lf→Mf→N→O→BRBR→Ls→Ms→BR It is important to resolve the issue and define precisely the steps in the photocycle(s?) in order to link the electrogenic steps of proton transport to specific transitions between known intermediates. In 1995, two of the current authors (R.I.S. and R.W.H.) concluded that both possibilities could be successfully mathematically modeled and that there was evidence that could be interpreted in favor of both (1). At the time, it appeared that perhaps both cooperativity and heterogeneity may be involved in the adaptation of the photocycle to actinic light. Now, almost 12 years later, in the face of additional information (briefly summarized above), and with newer, more extensive experimental data (included here) we are able to arrive at a more definitive resolution of the issue, namely, that a model based on heterogeneity is more likely to be true than one based on homogeneity and photocooperativity.