Zsolt Tokaji

Bleaching of bacteriorhodopsin by continuous light

A new two step photobleaching process is observed under continuous illumination of bacteriorhodopsin. This photobleaching is considerable even at physiological temperatures and becomes large at 50–60°C. The photobleaching also increases with increasing pH from 7 to 10. We suggest that the bleaching at its final stage could be due to the dissociation of the retinal and a local thermal denaturation‐like process. These facts may question the generally held belief that BR is a stable protein in vivo for a long period of time. Our results may have relevance also to practical applications of bacteriorhodopsin where the stability of bacteriorhodopsin is a key issue. In certain instances, the use of bacteriorhodopsin may require cooled conditions. Here, we defined the conditions under which bacteriorhodopsin is stable. The permanent photobleaching offers a new way of picture imaging and information input for bacteriorhodopsin‐based optical devices.

Actinic light density dependence of the bacteriorhodopsin protocycle

The photocycle of bacteriorhodopsin (BR) was studied in the 0.3 microsecond to 10 s time interval after excitation, using a wide range of actinic light intensities (10 ns half-duration, 0.06-60 mJ/cm2), at neutral and alkaline pH values. The relative weights of the rapidly and the slowly decaying components of the M intermediate (Mf and M(s), respectively) and the yield of the third millisecond component, N(R,P), are the function of the exciting light intensity (density), while their lifetimes are not. The relative weight of M(s) is found to be a linear function of the portion of the BR molecules undergoing the photocycle. This suggests the existence of a cooperative interaction of the BR molecules arranged in the crystalline purple membrane sheets. Another source of M(s) is also found, which results a nonvanishing relative weight of M(s) even at very weak actinic light density values. The explanation for this may be a branching, or the heterogeneity of BR itself or with its environment. It is shown that the relative weights of the rising and decaying components of the M form(s) do not correlate directly with each other.

Blue light regeneration of bacteriorhodopsin bleached by continuous light.

Photobleaching of bacteriorhodopsin (BR) by continuous light has recently been demonstrated. This bleaching consists of at least two subsequent product states. One of them is absorbing maximally in the blue spectral region. Our present study shows that upon illumination of the bleached sample with blue light a back photoprocess appears, resulting in regeneration of the original BR state. From a technical point of view, the observed phenomenon is similar to the reverting effect of blue light on the photocycle. An important difference is that the photobleached state of BR is much more stable than any of the photocycle intermediates, and may provide an advantage for several technical applications.

Cooperativity-regulated parallel pathways of the bacteriorhodopsin photocycle

The paper demonstrates that the actinic light density dependence of the millisecond part of the bacteriorhodopsin (BR) photocycle at high pH predicts a model, which is the same in the sequence of the intermediates as concluded previously on the basis of double flash experiments [1992, FEBS Lett. 311, 267–270]. This model consists of the Mf→N→BR and Ms→BR parallel pathways, the relative yields of which are regulated by cooperative interaction of the BR molecules. The decay of Ms is always slower than the decay of Mf and described as a direct reprotonation of the Schiff‐base from the bulk, and the recovery of the ground‐state nearly at the same time. Ms is decomposed into M′f and M′s. The first does not reprotonate, and similarly to Mf, it is suggested to be before the conformational change (switch), which latter process would be just before the decay of Mf. A simple way for the determination of the kinetics is also used. This confirms that the amount of N decreases with increasing fraction cycling and shows that the decay rate of N is independent of the fraction cycling. The differences in the kinetics are compared to each other, and they seem to allow a new way of kinetic evaluation at least under special conditions. The aim of this paper was briefly explained in my poster presented on the VIth International Conference on Retinal Proteins (see [14]).

Light-induced, long-lived perturbation of the photocycle of bacteriorhodopsin.

The relative weight of the slowly decaying M intermediate of the photocycle of bacteriorhodopsin increases upon increasing the energy density of the short (10 ns) actinic laser pulse. Moreover, when a pre‐exciting flash is applied to the BR sample, the absolute amplitude of the Ms is higher in the signal induced by a second flash, applied with a delay from 100 μs to 100 ms. These facts together prove that either the leftover BR ground‐state population becomes different due to the pre‐excitation, or there is a cooperative interaction between the BR molecules.

Kinetics of the N intermediate and the two pathways of recovery of the ground-state of bacteriorhodopsin

Absorption kinetic measurements at alkaline pH, in which bacteriorhodopsin (BR) is pre‐excited by another flash, indicate that a part of the recovery of the BR ground‐state is faster than the decay of the N intermediate of the photocycle. This fact proves the existence of a parallel pathway in the late part of the BR photocycle (the decay of Ms into BR), which does not include the N intermediate. We demonstrate that the decay of the Mr intermediate does not lead to any direct recovery of the BR ground‐state, and that excitation of N does not form an M‐like intermediate. Mr decays directly into the N intermediate, and photoexcitation of N leads to the formation of a red‐shifted form, O*. The kinetics of this red‐shifted intermediate are also presented.

Quantitative model for the cooperative interaction of the bacteriorhodopsin molecules in purple membranes

The trimeric, asymmetric and sequential model for the cooperative interaction of the bacteriorhodopsin molecules in purple membranes [Zs. Tokaji, Biophys. J. 65 (1993) 1130–1134] is being extended in the paper. Analyses of data from absorption kinetic measurements with preexcitation and green background illumination, and photoselection measurements on oriented samples confirm the main features of this cooperative interaction and support the validity of the extended model. This model includes the observed heterogeneity of the non‐excited state of the bacteriorhodopsin molecules in purple membranes, and the agreement with the data suggests that molecules in any other state than the bacteriorhodopsin ground state can alter the photocycle of their neighbors. The presented results seem to contradict other models for the cooperation of the bacteriorhodopsin molecules.

Light- and Redox-dependent Thermal Stability of the Reaction Center of the Photosynthetic Bacterium Rhodobacter sphaeroides¶

Abstract Irreversible loss of the photochemical activity and damage of the pigments (bacteriochlorophyll [Bchl] monomer, Bchl dimer [P] and bacteriopheophytin) by combined treatment with intense and continuous visible light and elevated temperature have been studied in a deoxygenated solution of reaction center (RC) protein from the nonsulfur purple photosynthetic bacterium Rhodobacter sphaeroides. Both the fraction of RC in the charge-separated redox state (P+Q−, where Q is a quinone electron acceptor) and the degradation of the pigments showed saturation as a function of increasing light intensity up to 400 mW cm−2 (488/515 nm) or 1100 μE m−2 s−1 (white light). The thermal denaturation curves of the RC in the P+Q− redox state demonstrated broadening and 10–20°C shift to lower temperature (after 30–90 min heat treatment) compared with those in the PQ redox state. Similar but less striking behavior was seen for RC of other redox states (P+Q and PQ−) generated either by light or by electrochemical treatment in the dark. These experiments suggest that it is not the intense light per se but the changes in the redox state of the protein that are responsible for the increased sensitivity to photo- and heat damage. The RC with a charge pair (P+Q−) is more vulnerable to elevated temperature than the RC with (P+Q or PQ−) or without (PQ) a single charge. To reveal both the thermodynamic and kinetic aspects of the denaturation, a simple three-state model of coupled reversible thermal and irreversible kinetic transitions is presented. These effects may have relevance to the heat stability of other redox proteins in bioenergetics.

Thermodynamics of Light-induced and Thermal Degradation of Bacteriochlorins in Reaction Center Protein of Photosynthetic Bacteria

Abstract The rate constants of thermal (irreversible) damage of bacteriochlorin pigments (bacteriochlorophyll monomer [B], bacteriochlorophyll dimer [P] and bacteriopheophytine [H]) in reaction center [RC] protein from the photosynthetic bacterium Rhodobacter sphaeroides were studied in the dark and during intense (400 mW·cm−2) laser light excitation (wavelengths 488 and 515 nm) under deoxygenated conditions. While the kinetics of degradation of P and B were monoexponential, the decay kinetics of H were overlapped by an initial lag phase at elevated (>40°C) temperature. This is explained by removal of the central metal ion from the bacteriochlorophylls as part of their degradation processes. At all temperatures, the rates of damage were very similar for all bacteriochlorin pigments and were larger in the light than in the dark. The logarithm of the rate constant of pigment degradation and loss of photochemistry as a function of reciprocal (absolute) temperature (Arrhenius/Eyring plot) showed single phase in the light and double phases in the dark. Below 20°C, the rate of pigment degradation in the RC decreased so dramatically in the dark that it became limited by the natural degradation process of bacteriochlorophyll measured in solution. The function of loss of photochemistry in the dark was also biphasic and had a break point at 40°C. The damage in the dark required high enthalpy change (ΔH‡ = 64 kcal/mol for P and ΔH‡ = 60 kcal/mol for B) and entropy increase (T·ΔS‡ = 38 kcal/mol for P and T·ΔS‡ = 34 kcal/mol for B at T = 300 K), whereas significantly smaller enthalpy change (ΔH‡ = 21 kcal/mol for P and B and ΔH‡ = 13 kcal/mol for H) and practically no (T·ΔS‡ = −1 kcal/mol for P and B at T = 300 K) or small (T·ΔS‡ = −9 kcal/mol for H at T = 300 K) entropy change was needed in the light. The thermodynamic parameters of activation reveal major steps common in the degradation of all bacteriochlorin pigments: ring opening reactions at C5 or C20 meso-bridges (or both) and breaking/removal of the phytyl chain. Their contribution in the degradation is probably reflected in the observed enthalpy/entropy compensation at an almost constant (ΔG‡ = 22–26 kcal/mol at T = 300 K) free energy change of activation.

N-like intermediate in the photocycle of the acid purple form of bacteriorhodopsin

© 1997 Federation of European Biochemical Societies.