IDENTIFICATION OF THE PROTON ACCEPTOR OF SCHIFF BASE DEPROTONATION IN BACTERIORHODOPSIN: A FOURIER-TRANSFORM-INFRARED STUDY OF THE MUTANT ASP85 → GLU IN ITS NATURAL LIPID ENVIRONMENT

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp . GRB, was investigated by static low-temperature and time-resolved infrared difference spec-troscopy. Measurements were also performed on the mutant protein labeled with [4-¹³C]Asp which allowed discrimination between Asp and Glu residues. 14,15-di¹³C-retinal was incorporated to distinguish amide-II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV-VIS measurements, our data show that from both the 540 and 610 nm species present in a pH-dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time-resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M-like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild-type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.     

THE RADIOLYTIC REDUCTION OF THE SCHIFF BASE IN BACTERIORHODOPSIN

Abstract —Nanosecond electron beam pulses cause radiolytic reduction of the retinal Schiff base in bacteriorhodopsin. The effects of different scavengers show that both H atoms and the formate radical anion can act as the reducing species. Model system studies on the radiolysis of retinal indicate transient formation of a retinal radical anion and suggest an analogous two-step reaction in bR. Delocalization of the radical in the polyene system and reaction with surrounding groups would account for the relatively low yield of reduced Schiff base and the appearance of intra- and intermolecular crosslinks in irradiated bacteriorhodopsin.     

EFFECTS OF MUTAGENETIC SUBSTITUTION OF PROLINES ON THE RATE OF DEPROTONATION and REPROTONATION OF THE SCHIFF BASE DURING THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions ofPro–50,Pro–91 andPro–186 on the deprotonation and reprotonation kinetics of the Schiffbase (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate thatPro–50 andPro–91, as well asPro–186, are important for the proton-pumping function of bR.     

THE EFFECT OF IONIC STRENGTH AND pH ON THE PROTONATED SCHIFF BASE AND TYROSINE DEPROTONATION KINETICS DURING THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— The deprotonation kinetics of tyrosine and the protonated Schiff base during the bacteriorho-dopsin photocycle were studied under different perturbations by transient absorption spectroscop Native purple membrane, as well as samples which were deionized (blue) then restored with Na⁺ or La³⁺ were used at pH's ranging from 7 to 10 at very low salt concentrations. The results were compared with previous studies at higher ionic strength. The important conclusions can be summarized as follows: (a) The rate constants of both the Schiff base and tyrosine deprotonation are not very sensitive to the changes of conditions. (b) An almost linear relationship is observed between the relative amplitudes of the tyrosine deprotonated during the cycle and the slow component of the Schiff base deprotonation under the different perturbations studied. This was taken to support the two site model for the protonated Schiff base, one near tyrosine and the other near its ionized form. (c) The pK_a value determined from the ratio of the amplitude of the fast to the slow component of the Schiff base deprotonation is found to decrease with increasing ionic strength of the medium. At extremely low ionic strength, it was found to equal that of the tyrosine phenolic group in solution.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP

Abstract
Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The p K values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP*

Abstract— Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The pK values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

A PROTON RELEASE SITE ON THE C-TERMINAL SIDE OF BACTERIORHODOPSIN

We have studied the pH dependence of the light-induced proton release and uptake by bacteriorhodopsin. The quantum efficiency of proton release in cell envelopes and proton uptake in phospholipid vesicles is high in the low pH range and begins to decline between pH 6 and 7 in cell envelopes and between pH 7–8 in phospholipid vesicles. In the cell envelope vesicles the proton release increases again above pH 8–8.5; in phospholipid vesicles a proton release is observed before proton uptake at pHs greater than 9. We suggest that the light-induced proton release observed at high pHs are due to protons released and rebound on the carboxyl terminal side of bacteriorhodopsin.     

BRANCHING IN THE BACTERIORHODOPSIN PHOTOCHEMICAL CYCLE

Abstract— Kinetics have been determined for the decay of the terminal phototransients M(410) and O(660) and the reappearance of the BR(570) chromophore in flash-photolyzed aqueous suspensions of light-adapted purple membrane fragments from Halobacterium halobium . The results were fitted to a linear model of     

TIME-RESOLVED RESONANCE RAMAN STUDIES ON THE PHOTOCHEMICAL CYCLE OF BACTERIORHODOPSIN

Abstract— Kinetic resonance Raman (RR) experiments were designed to study the time-behaviour of the retinal-binding protein bacteriorhodopsin (BR) in its photochemical cycle. The unphotolyzed chro-mophore B-570 and the two intermediates L -550 and M-412 were probed by the characteristic C=C stretching vibrations of the retinal moiety. Time resolution was achieved with a spinning cell as flow system in combination with two CW lasers in a pump-probe configuration. RR spectra were probed at 475 nm at various delay times between pump and probe event. The deconvolution of the spectra into the various components B-570, L-550 and M-412 was carried out by curve fitting procedures. It was found that at pH7.4 L-550 decays — with a time-constant of 62 μs — not completely but to a residual level of 35% of its initial value. This intermediate L -amplitude finally disappears in the ms-range (4.5 ms) synchroneously with the intermediate M -412. An analogeous time-behaviour was found at pH 4.6. In the basic range also an " L " -intermediate could be identified which is coupled to the long-lived M-component. To explain the peculiar time-dependence it is proposed that during the fast decay of L a dynamic equilibrium between L and M is established. Then during the reconstitution of B -570 the two intermediates disappear synchroneously. A molecular model is presented in which the dynamic equilibrium between L and M is explained by an oscillatory motion of a proton from the Schiff base group of the chromophore to its counterion.     

Schiff碱配合物的研究：Ⅶ.硅胶表面Schiff碱的配位作用

有机高分子Schiff碱对金属离子的螯合作用已引起人们的兴趣。硅胶为无定形高比表面无机高分子,通过氯甲基化,可进行多种化学反应而形成各种表面基团。本文报道四种新型硅胶表面Schiff碱(图1),考察了它们对Cu~(2 ),Co~(2 ),Cd~(2 )和Pb~(2 )的配位作用。     

FOURIER TRANSFORM INFRARED SPECTRAL STUDIES ON THE SCHIFF BASE MODE OF ALL-trans BACTERIORHODOPSIN and ITS PHOTOINTERMEDIATES,K and L*

Abstract– Difference Fourier transform infrared spectra were recorded for bacteriorhodopsin upon irradiation at 230, 170 or 77 K, which gave, respectively, the spectrum of the M, L or K intermediate minus unphotolyzed all-trans bacteriorhodopsin (denoted as BR). By replacement of the Schiff base nitrogen with ¹⁵N, or of either its hydrogen at N or C₁₅ with deuterium, the vibrational bands related to the Schiff base were identified and the isotope-shifts evaluated for BR, K and L. The 1348 cm^?l band of BR and K and the 1400 cm^?1 band of L were sensitive to each of these isotope substitutions. The 1254 cm^?1 band of BR, the 1245 cm^?1 band of K and the 1301 cm^?1 band of L were sensitive to either N- or C₁₅-deuteration but not to ¹⁵N-substitution. The N—D in-plane bending vibration of K and L appeared at 969 and 997 cm^?1, respectively, upon substitution with D₂O. All the results show that L is larger in frequencies related to the N—H in-plane bending vibration than K or BR and suggest that L has the strongest interaction with the protein. Among the bands containing an N—H bending vibration, the 1348 cm^?1 band of K was more intense than the corresponding band of L at 1400 cm^?1. The C₁₅-deuteration-induced upshift of the 1245 cm^?1 band of K was unobservable for the 1301 cm^?1 band of L. Such differences between L and K might be brought about by a distortion in the retinal moiety close to the protonated Schiff base of the 13-cis chromophore.     

THE PROTONATION OF A RETINYL SCHIFF BASE

The hydrogen bonding-protonation equilibrium for retinyl Schiff base/propionic acid or 3-chloropropionic acid systems was examined by Fourier transform infrared spectroscopy in non polar solutions at temperatures ranging from 25 degrees C to about -150 degrees C. The spectra give evidence for the gradual increase in the degree of protonation as temperature is lowered. The bearing of this on applying low temperature spectroscopic results to physiological conditions in rhodopsin research is discussed.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

ON THE ROLE OF TYROSINE IN THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract -The rate of formation of the M intermediate ( k _M) in the photocycles of bacteriorhodopsin (bR₅₇₀) and of nitrated bacteriorhodopsin (bR₅₃₂ⁿ), is measured over the range between pH 6.5 and 11.5. In the case of bR₅₇₀, k _M is markedly pH dependent, exhibiting a titration-like curve with pK ∽ 10.3. The pH dependency is completely eliminated by nitration. On the basis of previous work by Lemke and Oesterhelt (1981), the effect is attributed to the specific modification of the Tyr 26 residue. The data are rationalized by a mechanism in which deprotonation of Tyr 26 at the stage of the L intermediate constitutes a prerequisite for deprotonation of the retinal-lysine SchifT base. Both reactions are intimately associated with the photo-induced proton pump mechanism.     

STUDIES ON THE INTERACTION OF DNA AND WATER-SOLUBLE POLYMER-SUPPORTED SCHIFF BASE COMPLEX

The modes and activities of the interaction of DNA and water-soluble polymer supported Schiff base nickel complex, which has been prepared by copolymerization, have been discussed according to the fluorescent probe method. It indicates that the polymer matrix effect and increasing solubility in water can increase the interaction of this polymer metal complex with DNA.     

锰(Ⅱ)呋喃甲醛Schiff碱催化苯乙烯环氧化的研究

自1979年Groves首先以金属卟啉模拟细胞色素P－450,实现烯烃的环氧化^[1]以来,仿单加氧酶催化环氧化烯烃就成为仿酶催化领域里的一个非常活跃的研究课题^[2-5],但在这些报道中所用的模型化合物均为金属卟啉及其衍生物或Mn-Salen及其衍生物,这些化合物高昂的价格极大地限制了其应用前景。呋喃甲醛（俗称糠醛）取之于米糠或玉米芯,价格便宜且非石化产品,用它取代水杨醛不仅可降低成本,而且符合绿色化学要求。为此,本文选取了五种锰呋喃甲醛Schiff碱配合物作为模型化合物,以NaOCI为氧化剂,催化苯乙烯环氧化。讨论了配体结构、氧化物的pH值、轴配体、反应时间对催化环氧化反应的影响。     

Schiff碱配合物的研究——Ⅸ.表面Schiff碱配合物对H_2O_2分解的催化作用

制备了两种表面Schiff碱及其Cu~(2+)、Co~(2+)、Ni~(2+)、Zn~(2+)配合物,考察了它们对H_2O_2分解的催化性能,其活性顺序为:Co~(2+)>Cu~(2+)>Ni~(2+)>Zn~(2+),且与金属离子氧化还原电位有关。溶液的pH值增加有利于催化反应,有机配体的加入则对反应有所抑制。     

Schiff碱配合物的研究——Ⅸ.表面Schiff碱配合物对H2O2分解的催化作用

制备了两种表面Schiff碱及其Cu²⁺、Co²⁺、Ni²⁺、Zn²⁺配合物,考察了它们对H₂O₂分解的催化性能,其活性顺序为:Co²⁺>Cu²⁺>Ni²⁺>Zn²⁺,且与金属离子氧化还原电位有关。溶液的pH值增加有利于催化反应,有机配体的加入则对反应有所抑制。     

INVOLVEMENT OF THYLAKOID MEMBRANE-DEPENDENT PHOTOSENSITIZATION IN PHOTOINHIBITION OF THE CALVIN CYCLE ACTIVITY IN SPINACH CHLOROPLASTS

Photoinhibition of the light-regulated key enzymes of the photosynthetic carbon reduction (PCR) cycle was investigated using chloroplasts isolated from spinach leaves. Light quality dependence of the light-induced activity change (activation or inactivation) of key PCR enzymes in situ demonstrated that, while light activation is promoted mainly by red light (Λ.> 600 nm), inactivation takes place largely in the region of blue light (Λ < 500 nm). Inactivation was suppressed by a lipid soluble singlet oxygen (¹O_2,¹Δ_g) quencher. When “stromal protein” was subjected to a severe photoinhibitory treatment, no significant loss of activity was observed for any PCR enzyme assayed. However, the inclusion of thylakoids in the photolysis system resulted in a substantial inactivation of the enzymes; this inactivation was significantly diminished in the presence of imidazole and enhanced to some extent by a partial deuteration of medium. In contrast, superoxide dismutase did not exert any effect. The blue light-induced inactivation of the enzymes was remarkably decreased in the presence of thylakoids whose Fe-S centers were destroyed. The results obtained in this study suggest that photoinactivation of the PCR enzymes in situ is mediated mainly by ¹O₂, which is photoproduced primarily by the Fe-S centers of thylakoids and diffuses into the stroma.     

CONVERSION OF LIGHT ENERGY TO ELECTROSTATIC ENERGY IN THE PROTON PUMP OF HALOBACTERIUM HALOBIUM

Abstract— The energetics of the proton pump in Halobacterium halobium is considered on a molecular level. It is proposed that absorption of light by bacteriorhodopsin leads to charge separation and sequential changes in the proton affinities some of the protein groups resulting in proton pumping against the pH gradient. Two alternative models of 'through-space' and 'through-bond' charge separation are proposed. Novel diagrams that describe the energetics of the pumping process are presented. These diagrams can be used both to examine different models of bacteriorhodopsin and for considering other photobiological processes that involve conversion of light energy to electrostatic energy.