Proton FLASH Research Articles

Enhanced basicity of 1,3-dialkoxy-substituted benzenes: cyclophane derivatives

The photoprotonation of four dialkoxy-substituted benzenes in their excited singlet states has been studied. The parent systems 4 and 5 are regioselectively photoprotonated at the 2-position, with significant quantum efficiencies for deuterium incorporation at acidities greater than pH 2. The structurally related cyclophane derivatives 6 and 7 did not show any deuterium exchange over the same acidity range but fluorescence quenching by proton (in aqueous solution) and laser flash photolysis studies (in 1,1,1,3,3,3-hexafluoro-2-propanol) indicate that photoprotonation does take place with these compounds. Due to structural constraints imposed by the most stable conformation adopted by these molecules (6 and 7), the attacking proton (deuteron) is also the proton (deuteron) that is deprotonated from the cyclohexadienyl cation (2,6-dialkoxybenzenonium ion) intermediate, resulting in the absence of incorporation of deuterium.

Modification of carboxyl groups in bacteriorhodopsin. Chemical evidence for the involvement of aspartic acid residues in the structure and function of bacteriorhodopsin

Covalent modifications of the carboxyl residues of bacteriorhodopsin with α-diazo-p-nitro-acetophenone (1) under different conditions have been performed. The modified proteins have been characterized for their absorption, photochemical and proton pump activities. A partial characterization in terms of modification site has also been carried out. Three carboxyl residues of dark-adapted bacteriorhodopsin undergo reaction with 1 at pH 4.0, and the resulting protein exhibits absorption and proton pump activity similar to that of the native protein. Dark-adapted bacteriorhodopsin does not react with 1 at pH greater than 6.1. More than one carboxyl residues are modified when light-adapted bacteriorhodopsin is treated with 1 at acidic pH of 4.0 and 5.4. However, near physiological pH (7.2) only one carboxyl residue of light-adapted bacteriorhodopsin reacts with 1. These proteins exhibit absorption bands at 571 nm, fail to show proton translocation, and, upon flash photolysis, exhibit generation of ‘M’-like intermediates with τ1/2 of 13.47 ms, and λmax of 400 nm. The modified carboxyl residue is found to be located in the CNBr-9 (residues 72–118) fragment. Reaction of bacteriorhodopsin with 1 at –30 °C under photolytic (λ 500 nm) conditions at pH 7.2 results in the modification of two carboxyl residues, Asp-212 and another one in the CNBr-9 fragment. Such a modified protein exhibits drastically blue-shifted absorption at 400 nm, and does not show any proton translocation or flash photolytic activities. It has been concluded that during the photocycle at least two carboxyl residues exist predominantly in a protonated form. A molecular mechanism for the photocycle is also presented.

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron‐transport were estimated from the concentration of PSII reaction‐centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA320] and green [ΔA540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron‐transport were measured under the same light‐limiting conditions using 2,5‐dimethylbenzoquinone or 2,5‐dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSIIβ) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady‐state electron transport in isolated chloroplasts and caused underestimation of PSII electron‐transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash‐yield measurements. It is implied that quantitation of PSII by repetitive flash‐yield methods requires assessment of the slow turnover of electrons by PSIIβ and, in the presence of added quinones, assessment of the PSII quantum yield.