The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy. Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected. To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid. An incorporation of ca. 70% was obtained. The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate. Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed. The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway.

When pH-sensitive molecules are incorporated into liposomes, drugs can be specifically released from these vesicles by a change of pH in the ambient serum. Liposomes containing the pH-sensitive lipid palmitoyl homocysteine (PHC) were constructed so that the greatest pH differential (6.0 to 7.4) of drug release was obtained near physiological temperature. Such liposomes could be useful clinically if they enable drugs to be targeted to areas of the body in which pH is less than physiological, such as primary tumors and metastases or sites of inflammation and infection.

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca 2؉ -ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca 2؉ , the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P 3 -1-(2-nitro)phenylethyladenosine 5-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl 2 ؉ 330 mM KCl or 1 mM CaCl 2 ؉ 20% Me 2 SO): (i) formation of the ADP-sensitive phosphoenzyme (k app ‫؍‬ 0.79 s ؊1 ؎ 15% at 1°C, pH 7.0, 10 mM CaCl 2 , 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca 2؉ release (k app ‫؍‬ 0.092 s ؊1 ؎ 7% at 1°C, pH 7.0, 1 mM CaCl 2 , 20% Me 2 SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca 2؉ release. In 2 H 2 O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively.The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E 1 -E 2 model. -transport across the SR membrane to the hydrolysis of ATP. Its reaction cycle is shown in a simplified form in Fig. 1. Ca 2ϩ is bound from the cytoplasmic side of the membrane to high affinity binding sites of the ATPase (step on the left of Fig. 1), which enables the ATPase to use ATP as a substrate (1). Phosphorylation by ATP (upper step in Fig. 1) results in the occlusion of the bound Ca 2ϩ in the protein. The subsequent conversion of the phosphoenzyme from the ADP-sensitive to the ADP-insensitive form (step on the right of Fig. 1) leads to Ca 2ϩ release into the SR lumen. Hydrolytic cleavage of the phosphoenzyme completes the reaction cycle (bottom step in Fig. 1). An ATP molecule is shown bound to the ATPase throughout the cycle, which is the case at millimolar ATP concentrations (reviewed in Ref.2).The original model of the reaction cycle from deMeis and Vianna (3) was based on the assumption of two main functional states, E 1 and E 2 , of the protein. The interconversion between E 1 and E 2 is thought to be associated with a reorientation of the Ca 2ϩ -binding sites from the cytoplasmic s...

The protonation events that occur upon QA-QB-->QAQB- electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides were investigated by time-resolved infrared spectroscopy using tunable diode lasers as previously described [Mäntele, W., Hienerwadel, R., Lenz, F., Riedel, E. J., Grisar, R., & Tacke, M. (1990) Spectrosc. Int. 2, 29-35; Hienerwadel, R., Thibodeau, D. L., Lenz, F., Nabedryk, E., Breton, J., Kreutz, W., & Mäntele, W. (1992) Biochemistry 31, 5799-5808]. In the mid-infrared region between 1695 and 1780 cm-1, transient signals associated with QA-QB-->QAQB- electron transfer were observed and characterized. The dominant transient absorbance changes are three positive signals at 1732, 1725, and 1706 cm-1 and two negative signals at 1716 and at 1698 cm-1. The 1725 cm-1-signal disappears upon 1H-->2H exchange as expected for an accessible COOH group and is absent in Glu L212 Gln mutant reaction centers. On this basis, we propose an assignment of this signal to the COOH group of Glu L212. The other signals could correspond to intensity changes and/or shifts of other carboxylic residues, although contributions from ester C = O groups of bacteriopheophytins cannot be ruled out. In native reaction centers at pH 7 and at 4 degrees C, biphasic kinetics of the transient components were observed at most frequencies. The major signal at 1725 cm-1 exhibits a fast kinetic component of t 1/2 = 0.18 ms (25% of the total amplitude) and a slow one of t1/2 = 1 ms (75% of the total amplitude). A global fit analysis of the signals between 1695 and 1780 cm-1 revealed that the spectral distributions of the fast and the slow components are different. Biphasic kinetics with comparable half-times were also observed for the Glu L212 to Gln mutant. The simplest model to explain these results is that the fast phase represents electron transfer and the slow phase represents proton transfer and/or conformational changes coupled to electron transfer. The difference spectra of the slow component from native reaction centers show that the 1725 cm-1 band corresponds to an absorbance increase and not to a shift of an existing band. The signal is therefore proposed to arise from the protonation of Glu L212. The amplitude of the 1725 cm-1 signal varies distinctly with pH as expected for protonation of a COO- group. With increasing pH, the amplitude of the slow component increases while that of the fast component decreases slightly.(ABSTRACT TRUNCATED AT 400 WORDS)

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