循环光合磷酸化和光合作用

光合作用被称为"地球上最重要的化学反应",其二氧化碳同化是由还原辅酶II(NADPH)和腺三磷(ATP)来推动的。ATP主要来源于非循环光合磷酸化和循环光合磷酸化,但以往研究集中在前者。21世纪以来,随着测定技术的发展和多条与循环光合磷酸化有关的电子传递途径的发现,循环光合磷酸化的重要性和功能引起了极大地关注。该文作者结合自己实验室的相关的研究,围绕循环光合磷酸化的发现和重要性、同化力两个组分的比例与促进光合磷酸化提高光合作用的途径进行探讨,为进一步深入研究提供参考。     

The development of photosynthetic light reactions in cultured apple embryos in relation to their embryonal dormancy

The development of photosynthetic light reactions (photoreduction and photophosphorylation) was studied in cotyledons of cultured embryos isolated from stratified and non-stratified apple seeds (Malus domestica Borb. cv. Antonówka). The results obtained show the presence of an energy transfer inhibitor in the cotyledons of the stratified embryos.Abbreviations PSII photosystem II - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - ATP adenosine triphosphate - ADP adenosine diphosphate - CCCP carbonyl cyanide m-chlorophenyl-hydrazone - EDTA ethylenediamine tetraacetic acid; P, inorganic orthophosphate - TES (N-tris [hydroxymethyl] methyl-2-aminoethane sulfonic acid) This work was partly supported by the Polish Academy of Sciences, Contract No 10.2.10     

细胞色素C_3在Rhodopseudomonas caosulata载色体光合磷酸化中的作用

细胞色素C_3不仅能增进除去铁氧还蛋白载色体的循环光合磷酸化活性的恢复,而且也增进以DCPIPH_2为电子供体,维生素K_3、反丁烯二酸分别为电子受体构成的非循环光合磷酸化活性的恢复。 氩气相下,细胞色素C_3促进正常载色体光合磷酸化活性的最适浓度是1.8 μmol,当PMS存在时,这一促进作用随C_3浓度的增加而直线上升,然后呈稳态。 Antimycin A(10~(-7)mol/L)能充分抑制C_3参与的光合磷酸化活性,这一抑制现象在PMS存在时消失。 o~-phenanthroline(1×10~(-5)mol/L.)对C_3参与的光合磷酸化活性亦具抑制作用,并被PMS的添加而消失,当浓度提高时(10~(-3)mol/L),抑制现象不因PMS的存在而消失。 氢气相下的载色体光合磷酸化活性比氩气相下的低,并且随着载色体贮存(-20℃)时间的增长而急剧下降,24h贮存,丧失活性达60％。C_3对氢气相下的光合磷酸化活性具明显的促进作用,而铁氧还蛋白则不能,但两者同时存在时,其磷酸化活性显著提高。     

The coupling factor of photophosphorylation and the electric properties of the thylakoid membrane

The rate of ATP synthesis of illuminated chloroplasts is correlated with the electric conductance of their inner membranes. In agreement with previous studies it is shown that ATP synthesis is paralleled by an increased conductance of the thylakoid membrane. This conductance together with the ability to form ATP is abolished if chloroplasts are treated with an antibody against the coupling factor CF₁. It is not influenced by the fragmented monovalent antibody. This parallels the lack of influence of the fragmented antibody on ATP synthesis in contrast to its influence on hydrolysis and exchange reactions. We conclude that there are different sites for the interaction of the coupling factor with adenine nucleotides.Extraction of the coupling factor is shown to increase the membrane conductance by more than two orders of magnitude. Reincorporation of the crude coupling factor partially restores the net conductance of the membrane (increase in resistance by a factor of 2.5), while a higher degree of restoration was observed for ATP synthesis and the proton conductivity of the membrane. We conclude that the extraction procedure opens different conductive channels in the membrane; a proton specific one, possibly associated with the binding protein for the coupling factor, plus other channels for “non-protons” which in contrast to the proton channel cannot be plugged by reincorporation of the coupling factor.     

亚硫酸氢钠对光合机构及其运转的影响

NaHSO_2在不同的浓度范围内,可以影响光合、光呼吸、气孔开度和膜脂过氧化等,但在1～2mmol/L时常能提高净光合强度和增加作物产量。这可能与促进RuBP羧化酶/加氧酶活性及光合磷酸化有关。低浓度的NaHSO_3对RuBP羧化酶/加氧酶两种活性都有促进作用。0.1mmol/L NaHSO_3对弱光下测定的光合磷酸化稍有促进。NaHSO_3对植物的影响可能在某些方面与α-羟基磺酸类似,但有时也许能以其它形式影响植物,而不一定要变成α-羟基磺酸。     

The number of functional catalytic sites on F1-ATPases and the effects of quaternary structural asymmetry on their properties

Recent structural and kinetic studies of F₁ and F₀F₁ are reviewed with regard to their implications for the binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation. It is concluded that at least two and probably all three of the catalytic sites on F₁ are functionally equivalent despite permanent structural asymmetry in the soluble enzyme. A rotary mechanism in which all three catalytic subunits experience all possible interactions with the single-copy subunits during turnover is thought not to apply to soluble F₁ but remains an attractive model for the membrane bound enzyme.     

Gated proton conduction via the coupling factor of photophosphorylation modified by N,N-orthophenyldimaleimide

The membrane bound coupling factor of photophosphorylation is studied after pretreatment of broken chloroplasts with the bifunctional N,N-orthophenyldimaleimide under energization of the thylakoid membrane by mild flashing light. The proton conduction of the membrane is monitored both via the electrochromic absorption changes and via selective pH-indicating dyes. It is found that the coupling factor, after interaction with N,N-orthophenyldimaleimide during the preillumination period, shortcircuits one of the two protons pumped inside after excitation of chloroplasts with one short flash of light. In contrast to the low proton conductivity of the unperturbed thylakoid membrane (relaxation time for a proton gradient > 5s), this extra proton channel leads to a partial relaxation of a proton gradient within a few ms. Although limited to only one proton per electron, this extra proton conducting pathway is not otherwise specific. It operates with protons resulting from both Photosystem I and Photosystem II activity. In addition it operates with protons already present in the internal phase before firing of the exciting light flash. These effects are prevented by the presence of ATP (but not GTP) during the preillumination period. It is suggested that the modified coupling factor is gated open by the light induced electric field across the thylakoid membrane while self closing after passage of one proton per activated coupling factor.     

Influence of thylakoid protein phosphorylation on photosynthetic electron transport and photophosphorylation

Data are reported which show that thylakoid protein phosphorylation decreases photosystem II fluorescence yield and enhances the photosystem I dependent photophosphorylation catalyzed by phenazinemethosulphate in the presence of DCMU. The stimulation is larger at low light intensity, but is still observed at high intensity. These observations are interpreted to demonstrate that thylakoid protein phosphorylation causes a transfer of excitation energy from PS II to PS I, but may also have an independent stimulatory effect on PS I dependent photophosphorylation.     

叶片预照光对光合磷酸化和电子传递偶联效率的促进作用

菠菜叶片经预照光处理后提取的叶绿体,不但光合磷酸化活力增加,而且偶联效率P/O也提高,其提高P/O的作用与多粘菌素的作用不能叠加,叶片预照光可能通过使偶联因子产生漏能减少的变构而提高p/O的。用两阶段光合磷酸化法测得叶片预照光,在促进光合磷酸化时也能促进高能态积累。用CF_1抗体,NEM和五羟黄酮等处理叶绿体的研究表明,叶片预照光引起CF_1的构象变化——γ,亚单位上的SH基内埋而α.β亚单位上的催化中心却更加暴露。活体中的P/O值也许会比一般离体的高。     

Effects of organic solvents and tentoxin on enzyme-bound ATP synthesis in isolated chloroplast coupling factor 1

Isolated spinach CF1 (chloroplast coupling factor 1) forms enzyme-bound ATP without any supply of energy in the presence of high concentrations of Pi [Feldman and Sigman (1982) J Biol Chem 257: 1676-1683]. The final amount of CF1-bound ATP synthesized was increased greatly by 1,2-propanediol, and moderately by methanol, ethanol, and dimethyl sulfoxide, but decreased by glycerol and octyl glucoside. Methanol and ethanol greatly increased the initial rate of ATP synthesis, while 1,2-propanediol increased it only moderately. Low concentrations (10^-8 -10^-6 M) of tentoxin, which inhibit ATPase activity of isolated CF1, did not affect enzyme-bound ATP synthesis. However, high concentrations (>10^-5 M) of tentoxin, which stimulate ATPase activity of isolated CF1, enhanced the initial rate of CF1-bound ATP synthesis without significant effect on the final amount of ATP synthesized in the presence of medium ADP. The substrate of enzyme-bound ATP synthesized came largely from tightly bound ADP, not medium ADP, and tentoxin did not affect this substrate profile. Tentoxin did not affect the binding of medium ADP to high affinity sites on CF1.     

The role of ascorbate in the protection of thylakoids against photoinactivation

The inactivation of electron transport upon preillumination of isolated, stroma free thylakoids has been studied. Inactivation is defined here as the loss of activity which is not reversed upon relaxation of qE. It was found that both PS 2 and PS 1 dependent electron transport were inactivated, whilst the coupling of ATP synthesis to electron transport was not affected. The inactivation concerned both the transfer of excitation energy to the reaction centres, and the reaction centres themselves. Ascorbate protected against photoinactivation of the electron transport from H₂O to NADP or to methylviologen, much less the electron transport depending only on PS 1. The protection by ascorbate required its well known action as a cofactor of de-epoxidation of violaxanthin and the consequent formation of qE: under conditions where de-epoxidation was inhibited (presence of DTT or uncouplers) qE was also suppressed and ascorbate protection was abolished. Ascorbate did not p rotect the thylakoids against inactivation caused by H₂O₂in the dark.The latter was shown to concern mostly PS 2 electron transport.     

从二溴百里香醌的抑制效应分析融合膜电子传递的途径

甲基紫精(MV)系统中,在对类囊体膜的光合磷酸化(PSP)活力近于完全抑制的二溴百里香醌(DBMIB)浓度下,由类囊体残缺膜与线粒体嵴膜组成的融合膜PSP活力不仅不被抑制,反而受到不同程度的促进。在铁氰化钾(FeCy)系统中,DBMIB对类囊体膜的PSP活力不能完全抑制,同样浓度的DBMIB对融合膜的PSP活力有抑制效应。检测了不同膜在不同系统中,光下耗氧、放氧、FeCy还原和融合效应的关系等,论证了融合膜中电子传递的途径。     

Energy charge and emergence of the coleoptile and radicle at varying oxygen levels in Echinochloa crus-galli

The role of protein synthesis in senescence and in the inhibition of senescence by light and kinetin was studied in barley ( Hordeum vulgare L. cv. Hassan) leaves with different inhibitors of protein synthesis. A comparison of the actions of D- and L-chloramphenicol was made to compensate for the effects of D-chloramphenicol not mediated by inhibition of protein synthesis. The involvement of phytochrome was also studied. The results suggest that: 1) cytoplasmic protein synthesis is required for senescence in the light and in the dark; 2) chloroplasts, in the dark, synthesize protein which accelerates senescence; 3) kinetin inhibits the synthesis by chloroplasts of senescence-accelerating protein; 4) light changes the type of protein synthesized by chloroplasts from those accelerating to those retarding senescence; and 5) lightretar-dation of senescence is mediated by phytochrome and, probably, by photophos-phorylation.     

Energy-dependent changes in the ATP/ADP ratio at the tight nucleotide binding site of chloroplast ATP synthase

Using DTT-modulated thylakoid membranes we studied tight nucleotide binding and ATP content in bound nucleotides and in the reaction mixture during [¹⁴C] ADP photophosphorylation. The increasing light intensity caused an increase in the rate of [¹⁴C] ADP incorporation and a decrease in the steady-state level of tightly bound nucleotides. Within the light intensity range from 11 to 710 w m^–2, ATP content in bound nucleotides was larger than that in nucleotides of the reaction mixture; the most prominent difference was observed at low degrees of ADP phosphorylation. The increasing light intensity was accompanied by a significant increase of the relative ATP content in tightly bound nucleotides. The ratio between substrates and products formed at the tight nucleotide binding site during photophosphorylation was suggested to depend on the light-induced proton gradient across the thylakoid membrane.Abbreviations AdN adenine nucleotide - Chl chlorophyll - DTT dithiothreitol - FCCP carbonylcianide p-trifluoromethoxyphenilhydrazone - P_i inorganic orthophosphate - PMS phenazine methosulfate - TLC thin-layer chromatography - Tricine N-[tris(hydroxymethyl)methyl] glycine     

多粘菌素B对光合磷酸化的促进作用

多粘菌素B在低浓度的磷酸盐存在下,抑制光合磷酸化和电子传递,但在较高浓度的磷酸盐存在下,却能促进光合磷酸化和提高P/O值。多粘菌素B在促进光合磷酸化时与无机磷酸之间存在着类似竞争的关系,并显示出能增加叶绿体的毫秒延迟发光和用中性红表示的类囊体膜内外的pH变化,多粘菌素B亦能促进光下的ATP_P_j交换。根据上述实验结果;我们推测多粘菌素B在类囊体膜上可能有两个作用部位,一个在类囊体膜上,另一个在偶联因子(CF_1)的β亚单位上。     

低浓度尼日利亚菌素或氯化铵促进ATP形成机理的再探

在低盐介质中,含有垛叠类囊体（基粒）与不垛叠类囊体（间质片层）结构的叶绿体进行的光系统Ｉ电子传递与磷酸化反应（ＰＳＰ）都为低浓度的尼日利亚菌素所促进,低浓度的氯化铵对基粒结构叶绿体的系统Ｉ与包括两个系统的电子传递以及与之相偶联的磷酸化反应有促进效应,而对间质片层膜上进行的ＰＳＰ反应无影响。尼日利亚菌素或氯化铵对ＰＳＰ的促进效应在高盐介质中消失,且它们对高能态（Ｚ）形成的抑制效应在低盐介质中较高盐介质中大。当存在吡啶时,在低盐介质中它们对Ｚ形成的影响是抑制作用,在高盐介质中则是促进效应。这都表明不同结构的膜上均存在区域化质子。     

菠菜和蚕豆叶绿体CF_1结构与功能的比较

比较了菠菜和蚕豆叶绿体的光合磷酸化活力以及由不同活化方法活化的叶绿体及可溶CF1的Mg2＋－ATPase和Ca2＋－ATPase的活力,观测到两种叶绿体ATPase的合成和水解ATP的功能有明显差异。从两种叶绿体CF1的SDS－PAGE图谱上可见蚕豆CF1的ε亚基分子量明显小于菠菜的,蚕豆CF1的α和β亚基间分子量的差别也比菠菜的小。     

草甘膦对叶绿体能量转换的影响

除草剂草甘膦抑制植物叶绿体光合磷酸化活力,促进希尔反应活力,表现出明显的解偶联效应。它对叶绿体膜上腺三磷酶(ATPase)活力也起抑制效应,说明ATP合成被抑制不是由ATP酶活力变化所引起。这种解偶联现象主要是因光下质子转移受到抑制,在较低浓度的草甘膦影响下,先抑制质醌转移的质子进入膜内腔,浓度增加到20 mM,对水释放质子也有抑制。所以草甘膦对叶绿体能量转换的影响主要反映在质子转移被抑制,引起磷酸化活力受抑制。     

恒态光合磷酸化与△pH和△ψ的关系

在恒态下推动ATP形成的△μH~+中,除ApH外,△ψ的贡献也很明显:进行PSP反应,耗用H~+,降低了⊿pH幅度。NH_4Cl,尼日利亚菌素(Nig)对⊿pH和PSP的抑制作用是同步的。CCCP影响膜对质子的透性,加速内部质子外流,降低内部质子浓度,减少⊿pH幅度,抑制了PSP反应。PSP更依赖于类囊体内部质子浓度。在短杆菌环肽(Gram)(～5×10~(-8)mol/L)和缬氨霉素(Val)(～5×10~(-7)mol/L,+K~+)的作用下,⊿ψ几乎被完全消去,并对⊿pH无明显作用,但PSP分别被抑制了～30％(Gram)和～50％(Val),表明在恒态PSP反应中有30％～50％的能量来源可由⊿ψ提供。