Kinetics of photoconversion of protochlorophyllide 649 to chlorophyllide 676 at low temperature in etiolated cotyledons of Pharbitis nil

The kinetics of the photoconversion of protochlorophyllide 649 to chlorophyllide 676 were studied spectrophotometrically over the temperature range of ?15 – ?80°C under light-saturating conditions in etiolated cotyledons of Pharbitis nil. Photoconversion obeyed the sum of two first-order kinetics over this low temperature range. Activation energies obtained from the rate constants were about 5000 cal; this suggests that these two processes may be physical processes not chemical reactions. The results indicate that photoconversion involves two main steps. One is the step dependent on both light intensity and temperature that has been well studied. The other, which is concerned in this study, is the step dependent on temperature only, which may be the requisite for photoconversion. This latter step seems to be related to the binding mode of protochlorophyllide to a holochrome protein or to conformational changes in the protochlorophyllide-holochrome.     

Energy transfer from protochlorophyllide to chlorophyllide during photoconversion of etiolated bean holochrome

The photoconversion of protochlorophyllide to chlorophyllide in etiolated bean leaves or leaf extracts exhibits complicated kinetics that are neither simple first-order nor second-order with respect to the reactant. By comparing the chlorophyllide absorbance with the intensity of chlorophyllide fluorescence excited at wavelengths where both pigments absorb, we demonstrate that the kinetic complexity results from the transfer of electronic excitation from protochlorophyllide to chlorophyllide. Measurements of the polarization of chlorophyllide fluorescence indicate that efficient excitation transfer occurs at room temperature over pigment aggregates containing at least four molecules. The relative quantum efficiency of chlorophyllide-excited chlorophyllide fluorescence remains constant during photoconversion of holochrome or etioplast preparations. This result does not support the proposal of increasing exciton interaction between chlorophyllides during the course of photoconversion.     

Protochlorophyllide photoconversion mutants of Chlamydomonas reinhardtii

Summary We have developed a procedure for the isolation of Chlamydomonas reinhardtii mutants defective in light-dependent protochlorophyllide reduction (photoconversion), a key step in the biosynthesis of chlorophyll. Mutants were isolated by mutagenizing y-1-4, a temperature-sensitive yellow mutant blocked in the alternative light-independent protochlorophyllide reduction pathway, and screening for colonies which failed to green in the light at the restrictive temperature. Seven mutants were isolated which fail to photoconvert protochlorophyllide in photoconversion tests. All seven mutants have a single mutation at the pc-1 locus responsible for the defect in photoconversion. pc-1 maps close to y-5 on nuclear linkage group I. The pc-1 mutation is not itself temperature-sensitive because it blocks photoconversion at the permissive temperature when combined with the non-conditional yellow mutations y-5 and y-7. Cells containing the pc-1 mutation alone synthesize about 52% and 36% of the wildtype chlorophyll level in the dark and light, respectively, demonstrating that the light-independent protochlorophyllide reduction pathway in C. reinhardtii operates in the light.     

On the nature of the two pathways in chlorophyll formation from protochlorophyllide

The kinetics of formation of esterified chlorophyll in etiolated barley (Hordeum vulgare L.) leaves after illumination with a single flash was studied. It was found that after partial (14–24%) and after full photoreduction of protochlorophyllide, the same quantity of esterified products appear during the first 5 s after the flash. The rest of formed chlorophyllide was esterified in a slow process during at least 30 min at 15 °C. The product of fast esterification can be correlated with ‘short-wavelength’ chlorophyll, characterized by a fluorescence emission peak at 673–675 nm. This is the only chlorophyll form detectable within 20 s after partial (14%) photoconversion, and it appears at the same time as the shoulder of the chlorophyll(ide) fluorescence after full photoconversion. The main product after full photoconversion shows a fluorescence at 689 nm shifting in darkness within 15 s to 693 nm and then within 30 min to 682 nm (Shibata shift). The slow esterification proceeds with similar kinetics as the Shibata shift. We propose that the fast esterification of only part of total chlorophyllide after full photoconversion of protochlorophyllide in etiolated leaves reflects the restricted capacity of the esterifying system. The slow esterification of the residual chlorophyllide may be time-limited by its release from protochlorophyllide oxidoreductase, by disaggregation of prolamellar bodies and by diffusion of tetraprenyl diphosphates towards chlorophyll synthase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Circular dichroism studies on the structure and the photochemistry of protochlorophyllide and chlorophyllide holochrome

On the basis of absorption and circular dichroism (CD) spectral measurements, we conclude that the photoreduction of protochlorophyllide to chlorophyllide in homogenates of etiolated bean seedlings (Phaseolus vulgaris L.) involves two light steps in series. Before illumination, the active protochlorophyllide occurs in a dimeric form in the holochrome protein. The initial light reaction converts one of the protochlorophyllide molecules and forms a chlorophyllide-protochlorophyllide holochrome intermediate with a weak, characteristic CD spectrum. The second light reaction subsequently converts the second protochlorophyllide in a less efficient reaction that is temperature dependent. This produces a chlorophyllide holochrome which exhibits a strong double CD characteristic of dimers and which is stable below 1°C. At higher temperatures this dimeric chlorophyllide transforms in the dark to a monomeric form with low CD amplitude. Sucrose at high concentrations (2 M) alters the chlorophyllide holochrome CD spectrum and prevents the final dark dissociation step. Analysis of the photochemical kinetics confirms the occurrence of the two-step photoreduction and supports the stoichiometry of two (proto)chlorophyllides per holochrome protein.     

Activation volumes of processes linked to the phototransformation of protochlorophyllide determined by fluorescence spectroscopy at high pressure

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 degrees C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol(-1), which corresponds with the formation of one H-bond/molecule.     

Characterization of two phases of chlorophyll formation during greening of etiolated barley leaves

The esterification kinetics of chlorophyllide, obtained by a single flash of light, were investigated in etiolated barley ( Hordeum vulgare L.) and oat ( Avena sativa L.) leaves. A rapid phase, leading to esterification of 15% of total chlorophyllide within 15-30 s, was followed by a lag-phase of nearly 2 min and a subsequent main phase, leading to esterification of 85% of total chlorophyllide within 30-60 min. The presence of additional protochlorophyllide, produced in the leaves by incubation with 5-aminolevulinate, did not change the esterification kinetics. The rapid phase was identical after partial (11-15%) and full (>80%) photoconversion of protochlorophyllide; the ability for a second rapid esterification phase was restored in a dark period of at least 10 min. Cooling the leaves to 0 degrees C abolished the esterification of the main phase while the rapid phase remained unchanged. The prolamellar bodies were already in part transformed into prothylakoid-like structures within 2-5 min after a full flash but not after a weak flash (11% photoconversion); in the latter case, the corresponding transformation required a dark period of about 45 min. The existence of subcomplexes of prolamellar bodies containing NADPH:protochlorophyllide oxidoreductase and chlorophyll synthase in the ratio 7:1 is discussed.     

Chlorophyll Formation in Greening Bean Leaves during the Early Stages

In the evolution of the absorption spectrum of etiolated bean leaves (Phaseolus vulgaris L.) following illumination, a rapid photoconversion of 50% or more of the active protochlorophyllide at room temperature is followed by a shift of the chlorophyll(ide) absorption maximum: C₆₇₈→ →C₆₈₄→C_{672 nm}. Kinetic studies at 2 C and the absence of an isosbestic point provide evidence for an intermediate between C₆₇₈ and C₆₈₄. A dramatically different evolution is observed following the photoconversion of only 5 to 30% of the active protochlorophyllide at room temperature. C₆₇₂ appears within 30 seconds, and no subsequent dark shift occurs during the following 90 minutes. At 0 C, conversion of 5% of the active protochlorophyllide produces a new species, C₆₇₆, which converts progressively to C₆₇₂ within 10 minutes. We interpret the results in terms of two photochemical steps operating in series for the complete conversion of active protochlorophyllide. Furthermore, there appears to be competition between an irreversible, terminal dark shift and the second light reaction. We propose a scheme based on dimers of protochlorophyllide reduced stepwise to dimers of chlorophyllide in two successive light reactions. The intermediate mixed protochlorophyllide-chlorophyllide dimer absorbs at 676 nm and displays a much faster dissociation to monomers than does the chlorophyllide-chlorophyllide dimer.     

The Conversion of Protochlorophyllide636 to Protochlorophyllide630 in Leaves Treated with δ-Aminolevulinic Acid

Absorbancy changes in dark-grown, excised wheal leaves fed with δ-aminolevulinic acid are measured in vivo. The treatment with σ-aminolevulinic acid caused accumulation of protochlorophyllide, absorbing at 636 nm. After flashlight this form is found to convert in darkness to protochlorophyllide, absorbing at 650 nm. The conversion starts instantly after the leaves have been exposed to the flashlight, and the pre-existent pool of protocholorophyllidc absorbing at 650 nm will become emptied. The conversion is completed after 15–20 minutes, when a new pool of protochlorophyllide has been filled up. This new pool is transformed to chlorophyllide by a second flash and the sequence is repeated. The conversion may be composed of two reactions, a conclusion which can be drawn from the behaviour at different temperatures. One of these reactions is fairly temperature independent while the other is temperature dependent. The action of the protochlorophyllide holochrome is discussed.     

Photoconversion of protochlorophyll to chlorophyll a in a mutant of Chlorella regularis

Dark-grown cells of a mutant strain of Chlorella regularis containedchlorophyll a and protochlorophyll, phytyl ester of protochlorophyllide.Under illumination, protochlorophyll was quantitatively anddirectly converted into chlorophyll a. The photoconversion wasdependent on light intensity and temperature and proceeded ina cell-free preparation. The pathway of chlorophyll formation found in the mutant cellsis entirely different from that from protochlorophyllide byway of chlorophyllide a, which is generally observed in greenplants. ¹Present address: Division of Biology, Medical College of Miyazaki,Miyazaki 889-16, Japan. ²Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Ibaragi 300-21, Japan. (Received October 24, 1975; )     

Studies on the regeneration of protochlorophyllide after brief illumination of etiolated bean leaves

The effects of various inhibitors of nucleic acid and protein synthesis on protochlorophyllide synthesis in dark-grown Phaseolus vulgaris var. Red Kidney have been studied. Actinomycin D, chloramphenicol, and puromycin inhibit the regeneration of protochlorophyllide holochrome (detected as a 650 mμ absorption peak) in vivo in darkness after photoconversion of endogenous protochlorophyllide a to chlorophyllide a; this inhibition does not occur in similarly treated leaves supplied with δ-aminolevulinic acid.

These data suggest that the regeneration of protochlorophyllide results from the synthesis of RNA and enzymes required for the production of δ-aminolevulinate.

     

Nanosecond temperature jump relaxation dynamics of cyclic beta-hairpin peptides

The thermal unfolding of a series of 6-, 10-, and 14-mer cyclic beta-hairpin peptides was studied to gain insight into the mechanism of formation of this important secondary structure. The thermodynamics of the transition were characterized using temperature dependent Fourier transform infrared spectroscopy. Thermodynamic data were analyzed using a two-state model which indicates increasing cooperativity along the series. The relaxation kinetics of the peptides in response to a laser induced temperature jump were probed using time-resolved infrared spectroscopy. Single exponential relaxation kinetics were observed and fit with a two-state model. The folding rate determined for these cyclic peptides is accelerated by some two orders of magnitude over the rate of a linear peptide that forms a beta-hairpin. This observation supports the argument that the rate limiting step in the linear system is either stabilization of compact collapsed structures or rearrangement of collapsed structures over a barrier to achieve the native interstrand registry. Small activation energies for folding of these peptides obtained from an Arrhenius analysis of the rates imply a primarily entropic barrier, hence an organized transition state having specific stabilizing interactions.     

Light-Induced Breakdown of NADPH-Protochlorophyllide Oxidoreductase In Vitro

Light-induced loss of the enzyme protochlorophyllide reductase (EC 1.6.99.1.), already described as a characteristic of whole plants, has now been demonstrated in vitro using etioplast membrane preparations of Avena Sativa L. var Peniarth and Secale cereale L. var Rheidol. Some evidence is presented, based upon temperature, pH, and inhibitor sensitivity of the process, that loss of enzyme may be the result of proteolysis. The light-induced process can, in vitro, be largely prevented by addition of the substrates of the reductase, protochlorophyllide and NADPH. It is concluded that light causes the breakdown of the reductase in vivo and in vitro by producing ligand-free enzyme as a consequence of the photoconversion reaction.     

Protochlorophyllide oxidoreductase: "dark" reactions of a light-driven enzyme

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in the chlorophyll biosynthesis pathway. As POR is light activated, it allows intermediates in the reaction pathway to be observed by initiating catalysis with illumination at low temperatures, a technique that has recently been used to study the initial photochemistry. Here, we use low-temperature spectroscopy to show that the catalytic mechanism of POR involves two additional steps, which do not require light and have been termed the "dark" reactions. The first of these involves the conversion of the product of the initial light-driven reaction, a nonfluorescent radical species, into a new intermediate that has an absorbance maximum at 681 nm and a fluorescence peak at 684 nm. During the second dark step this species gradually blue shifts to yield the product, Chlide. The temperature dependence for each of these two processes was measured; the data revealed that these steps could only occur close to or above the "glass transition" temperature of proteins, suggesting that domain movements and/or reorganization of the protein are required for these stages of the catalytic mechanism.     

Glutaraldehyde fixation and protochlorophyll transformations in etiolated peas

Summary Glutaraldehyde causes protochlorophyllide and newly formed chlorophyllide in etiolated peas to absorb at shorter wavelemgths. Fixation completely inhibits photoconversion in a cell free preparation of protochlorophyll in 1 min but in intact infiltrated leaves inhibition is only partial after 24 hr. It is suggested that procedures which indicate that fixation has occurred at a broad level of structural organization may be inadequate to establish that fixation has occurred at a macromolecular level.     

Analysis of the subunit structure of protochlorophyllide holochrome by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The subunit structures of protochlorophyllide holochrome (PCH) and chlorophyllide holochrome (CH) were studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. PCH from leaves of dark-grown (Phaseolus vulgaris var. red kidney) is a polymeric pigment-protein complex of approximately 600,000 daltons. It is composed of 12 to 14 polypeptides of 45,000 daltons, when examined prior to and immediately following photoconversion. The protochlorophyllide or chlorophyllide pigment molecules are associated with these polypeptides. Subsequent to photoconversion, the absorption maximum of newly formed chlorophyllide shifts from 678 nm to 674 nm upon standing in darkness. Following the 678 to 674 spectral shift, the chlorophyllide is associated with a polypeptide with a molecular weight of 16,000 daltons. In addition, sucrose gradient centrifugation of PCH and CH under nondenaturing conditions indicates that during the course of the dark spectroscopic shift, the 600,000 dalton CH undergoes dissociation into a small chlorophyllide protein. The dissociation of CH, the change in the molecular weight of the chlorophyllide polypeptide from 45,000 to 16,000 daltons, as well as the dark spectroscopic shift are temperature-dependent and blocked below 0 C. It was also found that each holochrome molecule of 600,000 daltons contains at least four protochlorophyllide pigment molecules.     

Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.     

Beta-adrenergic modulation of cardiac ion channels. Differential temperature sensitivity of potassium and calcium currents

beta-Adrenergic stimulation of ventricular heart cells results in the enhancement of two important ion currents that regulate the plateau phase of the action potential: the delayed rectifier potassium channel current (IK) and L-type calcium channel current (ICa). The temperature dependence of beta-adrenergic modulation of these two currents was examined in patch-clamped guinea pig ventricular myocytes at various steps in the beta-receptor/cyclic AMP-dependent protein kinase pathway. External applications of isoproterenol and forskolin were used to activate the beta-receptor and the enzyme adenylate cyclase, respectively. Internal dialysis of cyclic 3',5'-adenosine monophosphate (cAMP) or the catalytic subunit of cAMP-dependent protein kinase (CS), as well as the external addition of 8-chlorphenylthio cAMP (CPT-cAMP) was applied to increase intracellular levels of cAMP and CS. Isoproterenol-mediated increases in IK, but not ICa, were found to be very temperature dependent over the range of 20-37 degrees C. At room temperature (20-22 degrees C) isoproterenol produced a large (threefold) enhancement of ICa but had no effect on IK. In contrast, at warmer temperatures (30-37 degrees C) both currents increased in the presence of this agonist and the kinetics of IK were slowed at -30 mV. A similar temperature sensitivity also existed after exposure to forskolin, CPT-cAMP, cAMP, and CS, suggesting that this temperature sensitivity of IK may arise at the channel protein level. Modulation of IK during each of these interventions was accompanied by a slowing in IK kinetics. Thus, regulation of cardiac potassium channels but not calcium channels involves a temperature-dependent step that occurs after activation of the catalytic subunit of cAMP-dependent protein kinase.     

Characterization of the photoconversion on reaction of the fluorescent protein Kaede on the single-molecule level

Fluorescent proteins are now widely used in fluorescence microscopy as genetic tags to any protein of interest. Recently, a new fluorescent protein, Kaede, was introduced, which exhibits an irreversible color shift from green to red fluorescence after photoactivation with lambda = 350-410 nm and, thus, allows for specific cellular tracking of proteins before and after exposure to the illumination light. In this work, the dynamics of this photoconversion reaction of Kaede are studied by fluorescence techniques based on single-molecule spectroscopy. By fluorescence correlation spectroscopy, fast flickering dynamics of the chromophore group were revealed. Although these dynamics on a submillisecond timescale were found to be dependent on pH for the green fluorescent Kaede chromophore, the flickering timescale of the photoconverted red chromophore was constant over a large pH range but varied with intensity of the 488-nm excitation light. These findings suggest a comprehensive reorganization of the chromophore and its close environment caused by the photoconversion reaction. To study the photoconversion in more detail, we introduced a novel experimental arrangement to perform continuous flow experiments on a single-molecule scale in a microfluidic channel. Here, the reaction in the flowing sample was induced by the focused light of a diode laser (lambda = 405 nm). Original and photoconverted Kaede protein were differentiated by subsequent excitation at lambda = 488 nm. By variation of flow rate and intensity of the initiating laser we found a reaction rate of 38.6 s(-1) for the complete photoconversion, which is much slower than the internal dynamics of the chromophores. No fluorescent intermediate states could be revealed.     

The two-step interaction between α-dimethylaminonaphthalene-1-sulfonyl-pepsinogen-(1–12) and pepsin

The binding between α-dimethylaminonaphthalenesulfonyl-(1–12) and porcine pepsin can be detected by the large changes that occur in the fluorescence spectra of the dimethylaminonaphthalenesulfonyl chromophore due to energy transfer from tryptophan residues of the protein. The interaction was previously shown to consist of two steps: a fast step leading to a greatly enhanced fluorescence followed by a slower rearrangement step which reduces the fluorescence but leads to tighter binding and inhibition of the catalytic activity of pepsin (1). The two steps have been studied over a wide range of values of pH, temperature, and ionic strength to gain additional insights into the physical events occurring during the interaction. Based on the pH and ionic strength dependence, the initial step most likely involves electrostatic interaction of the basic peptide inhibitor with the acidic surface of pepsin in a rapid collision process. The use of this fluorescent reporter group has also suggested that the equilibrium binding after the slower rearrangement may also be pH dependent with most effective binding at higher pH. The kinetics of the slow step were measured by monitoring the continuous fluorescence decay. The resulting rates are compared to the rates observed by others for binding of pepstatin to pepsin. From the pH dependence of fluorescence, pK_app values are obtained for the dansylated peptide (3.25), for the pH dependence of the initial binding step (4.87), and for the equilibrium position (4.75).