THE KINETICS OF THE EARLY STAGES OF THE PHYTOCHROME PHOTOTRANSFORMATION Pr→ Pfr. A COMPARATIVE STUDY OF SMALL (60 kDalton) and NATIVE (124 kDalton) PHYTOCHROMES FROM OAT

A comparative study of the decay kinetics of photogenerated transients from small (60 kDalton) and native (124 kDalton) oat phytochrome in the red-absorbing form (P_r) in phosphate buffer containing 5 mM ethylenediamine tetraacetic acid, pH 7.8, (PB) and in PB containing 20% ethylene glycol, has been carried out in the temperature range 275–298 K. The analysis confirmed that at least two primary photoproducts, intermediates Iⁱ₇₀₀s and Iⁱ_7oo are formed from P_r. The kinetic parameters, as observed in PB at 695 nm and 275 K, are similar for the I₇₀₀ intermediates of both small and native phytochrome. Namely, the lifetimes are about 21 μs (component percentages 38%) for the I Iⁱ₇₀₀s and about 200 μ.s (62%) for the Iⁱ₇₀₀S- Arrhenius preexponential factors (A) of about 10¹⁶ and 10¹⁵ s-¹and activation energies of about 61 and 56 kJ/mol were measured for the absorbance decays of the I₇₀₀S of small and native phytochrome, respectively. The kinetic data favour parallel paths for the formation of the Iⁱ₇₀₀s from P_r, and the activation parameters indicate that the primary photoreactions of the transformation from P_r to the far-red-absorbing form are restricted to the chromophore within the protein. Moreover, the relatively modest temperature dependence of the lifetimes of the Iⁱ₇₀₀^S from small and native P_r supports the working hypothesis that the ground state reactions to the Iⁱ_bl, intermediates–although somewhat influenced by the polypeptide fragment that is removed upon degradation of native to small P_r–are localized to the chromophore, as is most probably the case also for the primary photoreactions. The effect of the addition of 20% ethylene glycol on the pre-exponential factors of the time-dependent decay functions is discussed in similar terms of the early stages of the phototransformation.     

PHOTOACTIVITY OF TRANSIENT INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF Avena PHYTOCHROME AS OBSERVED BY A DOUBLE-FLASH TRANSIENT-SPECTRUM ANALYZER

Abstract— Phototransformation from the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form occurs via at least two reaction stages. We report here on the photoactivity of the intermediates present during these two stages, as detected with 114/118-kdalton Avena phytochrome by a transient spectrum analyzer with two actinic lasers of microsecond duration. Flash activation of intermediates present during the first stage results in their photoconversion back to Pr within the time resolution provided by the analyzer, which is 10 μs. Since these intermediates are present within 60 ns of excitation of Pr (Cordonnier et al. , 1981), a single flash of ms duration as used here should yield a photostationary equilibrium between them and Pr. The proportion of Pr converted to intermediates by a single saturating flash is estimated to be about 30%. Flash activation of intermediates present during the second stage converts them to another transient intermediate stage, which decays thermally to Pr within 2 ms.     

ANALYSIS OF PHOTOTRANSFORMATION INTERMEDIATES IN THE PATHWAY FROM THE RED-ABSORBING TO THE FAR-RED-ABSORBING FORM OF AVENA PHYTOCHROME BY A MULTICHANNEL TRANSIENT SPECTRUM ANALYZER

Phototransformation of the red-absorbing form of phytochrome (Pr) to the far-red-absorbing form (Pfr) was followed with a custom-built transient spectrum analyzer. Large phytochrome, which consisted of approximately 120000-dalton monomers, was immunopurified or conventionally purified from etiolated oat (Avena sativa L., cv. Garry) shoots. Phototransformation was initiated by exciting Pr with a 115-mJ, 600-ns half-width, 655-nm laser pulse. Absorption spectra were recorded on a microsecond time scale at predetermined times after the flash. It has been reported earlier that flash excitation of large oat Pr produces a transformation intermediate with maximum absorbance near 700 nm in a difference spectrum and that this intermediate decays by two kinetically distinct reactions. Difference spectra for these two reactions are indistinguishable. Both show bleaching centered at 690 nm with no detectable associated absorbance increase between 570 and 830 nm. Subsequent appearance of absorbance at 724 nm, which presumably but not necessarily represents the appearance of Pfr, had earlier been shown to occur by two kinetically distinct reactions for large oat phytochrome. Data presented here indicate in addition the occurrence of a third, slower reaction. Difference spectra for the two faster reactions are indistinguishable, both with maxima near 728 nm and minima near 650 nm. The difference spectrum for the slowest component, however, was qualitatively different exhibiting a maximum near 722 nm with no corresponding minimum. About 15-20% of the absorbance increase at 724 nm occurred by this slowest reaction, which exhibited a half-life of 3 s at 25°C and a Q₁₀ of 1.2 for immunopurified and 1.5 for conventionally purified phytochrome. The percentage occurring by this reaction was independent of temperature over the range studied (1-25dEC). For immunopurified phytochrome the enthalpy of activation, Gibbs free energy of activation, and entropy of activation of this slowest reaction were found to be about lOkJ-mol^-1, 75kJ.mol^-1, and -220 J.mol^-1 K^-1, respectively, and for conventionally purified phytochrome 25kJ.mol^-1, 75kJ.mol^-1and —170 J.mol^-1 K^-1, respectively. The thermodynamic characteristics of this reaction indicate that it may involve a significant ordering of the protein moiety as it transforms to Pfr.     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris-I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND-LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix-mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far-red-absorbing form of this pigment, P_fr, was investigated with saturating irradiations between 662 and 747 nm under low-fluence-rate conditions. These control irradiations establish a proportion of the total phytochrome, P,_tot, as P_fr with P_fr/P_tot–φ at equilibrium. These φ -values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix-mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye-laser pulses of 380 ± 30 ns under high-fluence-rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red-absorbing form of phytochrome, P_fr on the final P_fr-level. Spore germination was initiated by a single-laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix-mas. Both saturation levels correspond to a ø-value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high-fluence-rate conditions during the laser pulse which establishes the photochromic system between P_r and a set of very early intermediates, Iⁱ_700, (= P_r? Iⁱ₇₀₀). This system can be described by the extinction coefficients of P_r and the intermediates Iⁱ₇₀₀, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of P_r and Iⁱ_7(l() (= lumi-R) given by Eilfeld and Riidiger (1985, Z. Naturforsch. 40c , 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φ_pr-φ1700: φ_1700φpr=1:1 and an assumed additional dark reaction leading from Iⁱ₇₀₀ or later intermediates back to P_r: or (ii) with a quantum yield ratio φ_{pr φ 1700}: φ_{1700 φpr}=1:2. In this case all Iⁱ₇₀₀ have to relax to P_fr. In this case all Iⁱ₇₀₀ have to relax to P_fr.     

A PHYTOCHROME PHOTOTRANSFORMATION STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: ANALYSIS OF THE DECAY MECHANISM OF I700*

The mechanism of I₇₀₀ decay, representing an early event in the phytochrome P_r→ P_fr phototransformation, was reanalyzed in the microsecond range by conventional laser flash photolysis as well as by two-laser/two-color flash photolysis. Three kinetic models that might describe the I₇₀₀ decay mechanism following P_r excitation were considered: a parallel, a sequential, and an equilibrium model. These models were used to mathematically simulate both the one- and two-laser flash experiments in an effort to select the model best describing the I₇₀₀ decay. The sequential model could be excluded already on the basis of the one-laser flash photolysis results alone. Discussion of the two-laser/two-color flash rcsults in the context of the equilibrium and the parallel models is presented.     

PICOSECOND DYNAMICS OF THE EXCITED STATE RELAXATIONS IN PHYTOCHROME

Abstract— A comparative study of the picosecond kinetics of rye ( Secale cereale L.) phytochrome, its 39 and 23 kDa chromopeptides and deuterated rye phytochrome has been carried out. Evidence is presented that the fluorescence decay of Pr contains a very short lifetime component (14 ps) which has escaped detection in the fluorescence studies reported so far. Thus, the overall decay is well described by four exponential components, two rapid (14 and 44 ps) and two slower ones (157 and 690 ps). The fluorescence decays of deuterated Pr and of a 39 and 23 kDa chromopeptide of Pr also require the analysis in terms of four exponentials for a good fit. Some of the lifetime and amplitude values obtained differ significantly from the values estimated for Pr. In the chromopeptides, the two longer components have distinctly slower decays. For the two faster components the lifetimes remain approximately the same, but their relative amplitudes vary greatly. In deuterated Pr, the lifetimes are affected only slightly by deuteration. In contrast, the decay amplitudes are strikingly altered. Moreover, from a rate equation simulation modelling the observed fluorescence kinetics, it turns out that the yields for the various deactivation steps in the chromopeptides and in deuterated Pr reveal differences from the corresponding values in Pr. The implications of the results presented with respect to the influence of the protein moiety of Pr on the picosecond relaxation process are discussed.     

DIRECT MEASUREMENT OF PHYTOCHROME PHOTOCONVERSION INTERMEDIATES AT HIGH PHOTON FLUENCE RATES

A custom-built modulated split-beam spectrophotometer has been used to measure the absorbance of tissue samples and purified phytochrome whilst exposing the sample to actinic 633 nm laser radiation at fluence rates approaching those of daylight. This approach has allowed the direct observation of the accumulation of phytochrome photoconversion intermediates at high fluence rates. At ca 1250 μmol m^?2 s^?1 upwards of 35% of the total phytochrome was present in the form of photoconversion intermediates in tissues of maize, sunflower and tomato. In other tissues tested (wheat, bean and Amaranthus) and in purified oat phytochrome, rather smaller levels of intermediates accumulated. Upon “lights-off” only a proportion of the accumulated intermediates decayed to far-red absorbing phytochrome (Pfr), the remainder appearing as the red-absorbing form (Pr). Difference spectra suggested that, at high light levels, Pr may be reformed via a photochemical back-conversion of an intermediate in the Pr—Pfr pathway, although the involvement of intermediates in the Pfr—Pr pathway cannot be excluded. The implications of the results for the ecological function of phytochrome are discussed.     

KINETICS OF Pfr APPEARANCE IN Amaranthus caudatus‡

Abstract— The kinetics of the far-red absorbing form of phytochrome (P_fr) appearance from intermediates in the pathway from the red absorbing form of phytochrome (P_r) to P_fr that accumulate under high fluence rate white light have been investigated in 3-day old dark grown Amaranthus caudatus seedlings. The appearance of P_(r after a 5 s white light pulse was measured over the temperature range -8 to 25^°C in samples flushed with O₂ or N₂. Over the whole temperature range under anaerobic conditions the kinetics of the slowest component of P_fr appearance are faster than in the presence of O₂. Arrhenius plots are linear over this temperature range and indicate the activation energy for the slowest component of P_fr appearance is 44.05 ± 1.97 kJ mol^?1 for O₂ and 53.69 ± 4.86 kJ mol^?1 for N₂.     

EVIDENCE FOR THE EXISTENCE OF MEMBRANE-ASSOCIATED PHYTOCHROME IN THE CELL

Abstract Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were camed out. The membrane fractions prepared in the absence of Mg²⁺ from etiolated coleoptiles contained 13% of total photoreversible phytochrome, which was readily solubilized by mild detergents. Its molecular size was indistinguishable from soluble phytochrome and equal to nondegraded maize phytochrome. Low-temperature fluorescence studies with intact tissue found that the position of the emission maximum at 85 K (λ_max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (γ₁), varied in different parts of etiolated seedlings: λ_max and γ₁ reached their maximum values in the tips of coleoptiles and roots, 686 nm and 0.30–0.40, whereas the lowest values, 682 nm and ca 0.05, were observed in the root base. These parameters correlated well with those obtained for the pigment in the soluble and membrane-containing fractions: 684 and 680 nm, and 0.33 and 0.06, respectively. The extent of the Pr phototransformation into the far red-absorbing form (Pfr) (γ₂) did not differ much: values of 0.80–0.85 and 0.70–0.75 correlated with the high and low values of γ₁. These variations of the parameters were interpreted in agreement with our previous observations in terms of two phytochrome A species whose relative concentrations vary depending on the experimental conditions—the longer wavelength bulk light-labile species with high γ₁ (Pr″), and the shorter wavelength minor light-stable species with low γ₁ (Pr″). Close similarity between Pr’and the soluble phytochrome and between Pr″ and the membrane-bound phytochrome points to the possible origin of the native Pr’and PrPrime; species, thus providing evidence for the existence of membrane-bound pigment in the cell.     

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris–II. In vivo KINETICS OF THE DECAY OF Ii700 AND OF THE FORMATION OF Pfr STUDIED WITH A DOUBLE-LASER APPARATUS*

Abstract— Photoconversion of the red-light-absorbing form of phytochrome, P_r, to the far-red-light-absorbing form, P_fr, was investigated in vivo at 22°C with 600 or 800 ns laser pulses of high spectral purity and induction of spore germination in Dryopteris paleacea was used as indicator for the progress of photoconversion. This reaction is initiated by a saturating R-laser pulse of 648.5 nm, establishing an equilibrium of the photochromic system between P_r and the very early intermediates, Iⁱ₇₀₀ (P_rφ Iⁱ₇₀₀)- The decay of Iⁱ₇₀₀ as well as the formation of P_fr was recorded by the application of a second pulse varied between 698 and 717.5 nm, which inhibits the formation of P_lr being absorbed predominantly by Iⁱ₇₀₀or P_fr, respectively. The most effective inhibition for the second pulse is found up to 10 u.s after the first pulse and this is interpreted by photoreversion of Iⁱ₇₀₀ to P_r; thus reducing the formation of P_fr from Iⁱ₇₀₀. This early inhibition decreases between 10 μs to 100 ms after the R-laser pulse, as a result of the decay of Iⁱ_bl to a bleached species I,;. This decay can be described by three first order kinetics with the rate constants k₁₂= 16830 ± 2970 s^-1, k₁₂= 666 ± 218 s^-1,k₁₃= 9.8 ± 0.9 s^-1. A second inhibition, due to the formation of P_fr, is found for dark intervals <100 ms and can be described by two first order kinetics with the rate constants k₂₁= 2.9 ± 0.6 s^-1 and k₂₂= 0.17 s^-l.     

Temperature dependence of small polaron population decays in iron-doped lithium niobate by Monte Carlo simulations

The population decay of light-induced small polarons in iron-doped lithium niobate is simulated by a Monte-Carlo method on the basis of Holstein's theory. The model considers random walks of both bound polarons (Nb_Li⁴⁺) and free polarons (Nb_Nb⁴⁺) ending to deep traps (Fe_Li³⁺). The thermokinetic interplay between polaron species is introduced by trapping and de-trapping rates at niobium antisites (Nb_Li). The decay of the Nb_Li⁴⁺ population proceeds by three possible channels: direct trapping at Fe_Li³⁺ sites, hopping on niobium antisites and hopping on Nb regular sites after conversion to the free state. Up to three regimes, each one reflecting the predominance of one of these processes, appear with different activation energies in the Arrhenius plots of the decay time. The influence of Fe_Li and Nb_Li concentrations on the transition temperatures is evidenced. For both polaron species, the length of the final hop (trapping length) is found much larger than the usual hopping length and decreases at rising temperature. This trap size effect is a natural consequence of Holstein's theory and may explain some unclear features of polaron-related light-induced phenomena, such as the temperature-dependent stretching exponent of light-induced absorption decays and the anomalous increase of the photoconductivity at high doping levels.     

KINETICS OF PHYTOCHROME PELLETABILITY*

Abstract— The pelletability of P_r from maize coleoptiles was studied as a function of the delay time between a red and a far-red light pulse given in vivo. The obtained curve can be resolved into three parts. The two slowest reactions have half lives of 40 s and 3.6 min at 0°C. Furthermore, a break in the Arrhenius plot from the slowest reaction of the curve indicates that either the phytochrome “receptor” or the phytochrome molecule itself undergoes a jump in the Arrhenius activation energy at 20°C. These data are in good agreement with kinetic studies of phytochrome pelletability also discussed in this paper.     

PHOSPHORESCENCE QUENCHING OF POLY rA IN GLUCOSE-SODIUM ACETATE SOLUTION AT 77 K*

Abstract. The phosphorescence of poly rA at 77 K in an aqueous medium containing glucose is half-quenched by bound ethidium bromide at a fractional concentration, r_1/2, of 0.005, and by bound proflavine at r_1/2= 0.002: r_1/2= 0.024 for Co²⁺ and r_1/2= 0.039 for Mn²⁺. The decay of the dye-quenched poly rA phosphorescence is markedly nonexponential and decays more rapidly with increasing dye concentration, while the decay of the metal-ion quenched poly rA phosphorescence parallels that of the unquenched poly rA, independent of metal-ion concentration. Förster overlap integrals and critical distances for transfer of both poly rA singlet and triplet excitation to dye singlet states are calculated, and used to explain a consistent interpretation of the experimental results in terms of one-step direct excitation transfer from base to dye or metal ion in a highly folded polymer conformation.     

Estimation of the normal boiling points of water and 1,2-dimethoxyethane through the viscosity–temperature dependence in the corresponding binary mixtures

Excess quantities calculated from literature values of experimental density and viscosity in 1,2-dimethoxyethane + water binary systems (from 303.15 to 323.15 K) can lead us to test different correlation equations as well as their corresponding derivative properties. Inspection of the Arrhenius activation energy (Ea) and the enthalpy of activation of viscous flow (ΔH*) shows very close values; here, we can define partial molar activation energies Ea₁ and Ea₂ for 1,2-dimethoxyethane and water, respectively, along with their individual contribution separately. Correlation between the two Arrhenius parameters of viscosity in all compositions shows existence of main distinct interaction behaviours delimited by particular mole fractions in 1,2-dimethoxyethane. Moreover, we add that correlation between Arrhenius parameters reveals interesting Arrhenius temperature which is closely related to the vapourisation temperature in the liquid vapour equilibrium, and the limiting corresponding partial molar properties can permit us to estimate the boiling points of the pure components.     

Flavinium Catalysed Photooxidation: Detection and Characterization of Elusive Peroxyflavinium Intermediates

Flavin‐based catalysts are photoactive in the visible range which makes them useful in biology and chemistry. Herein, we present electrospray‐ionization mass‐spectrometry detection of short‐lived intermediates in photooxidation of toluene catalysed by flavinium ions (Fl⁺). Previous studies have shown that photoexcited flavins react with aromates by proton‐coupled electron transfer (PCET) on the microsecond time scale. For Fl⁺, PCET leads to FlH^.+ with the H‐atom bound to the N5 position. We show that the reaction continues by coupling between FlH^.+ and hydroperoxy or benzylperoxy radicals at the C4a position of FlH^.+. These results demonstrate that the N5‐blocking effect reported for alkylated flavins is also active after PCET in these photocatalytic reactions. Structures of all intermediates were fully characterised by isotopic labelling and by photodissociation spectroscopy. These tools provide a new way to study reaction intermediates in the sub‐second time range.     

THE EFFECT OF DEUTERIUM OXIDE ON THE FLUORESCENCE AND PHOTOTRANSFORMATION OF 124 kDALTON PHYTOCHROME*

Abstract— To probe the nature of primary photoprocess and the mechanism of the phototransformation of undegraded 124 kDa oat phytochrome, solvent deuterium isotope effects on the fluorescence and phototransformation of phytochrome have been investigated. The fluorescence intensity and lifetime of phytochrome (P_r form) are greater in D₂0-buffer than in H₂O-buffer, suggesting a possible involvement of proton transfer in the primary photoprocess of phytochrome. Although the photostationary equilibrium (P_r to P_fr ratio) was not altered by deuterium oxide, in contrast to degraded phytochrome, the rate constants of both transformations, P_r→ P_fr and P_fr→ P_r were enhanced by up to 24%. The P_r to P_fr phototransformation of degraded phytochrome, however, was retarded by about the same percentage in D₂O. These opposite effects of D₂O with degraded and undegraded phytochromes underscore the fact that the P_r form from the former reverts to the P_r form in the dark, apparently catalyzed by deuterated general and/or conjugate acidic group(s). With the degraded phytochrome the deuterium oxide enhancement of the rate of dark reversion was approximately 2-fold (Sarkar and Song, 1981). Both the fluorescence intensity and the rates of phototransformation of phytochrome were enhanced in D₂O with successive photocyclings (P_r→ P_fr→ P_r→ P_fr→ P_r etc.) with alternating red and far-red irradiation. It has been proposed that successive photocycling of phytochrome in D₂O results in proton-deuteron exchange in the partially exposed P_tr chromophore and/or its surrounding amino acid residues.     

CHARACTERIZATION OF A MICROSECOND INTERMEDIATE IN THE LASER FLASH PHOTOLYSIS OF SMALL PHYTOCHROME FROM OAT

Abstract— Irradiation of small phytochrome from oat in its P_r form with 15 ns laser pulses of different wavelengths(605–655 nm) gave rise to a difference absorption with maxima at 400 and 685 nm for the first detectable transient. Bleaching of a 660 nm band was observed, non-recuperable up to 1 ms. The transient absorption has a lifetime of 70±15 μs at 273 K. The transient is tentatively identified as lumi-R and the conformation of its chromophore is postulated to be more extended than that of P_r. A deviation from the exponential decay of the lumi-R absorption at 284 and 300 K and the lack of observable enhancement of the far-red absorption within 1 ms are interpreted in terms of the appearance of still other intermediates on this time scale between lumi-R and P_fr phytochrome.     

PHYTOCHROME CONTROL OF ITS OWN SYNTHESIS IN Sorghum vulgare AND Avena sativa

The accumulation of phytochrome in the dark was measured for Avena sativa seedlings after a white light pretreatment and for Sorghum vulgare seedlings after continuous red or far-red light treatments, using the herbicide Norflurazon to prevent greening under continuous irradiation. In both cases the accumulation of phytochrome depends on the state of the phytochrome at the light-dark transition: high P_fr levels (red light pulse) led to a slower rate of phytochrome accumulation than lower P_fr levels (long wavelength far-red (RG 9) light pulse). Poly-(A⁺)-RNA was isolated fromA. sativa seedlings grown for 48 h in darkness + 24 h WL + light pulse (5 min) (red, RG 9 light, red followed by RG 9 light or RG 9 followed by red light pulse) + 19 h darkness. The poly-(A⁺)-RNA was translated in a rabbit reticulocyte lysate system and the translation products were immunoprecipitated by specific anti-phytochrome antibodies. It was demonstrated that the activity of mRNA coding for phytochrome was under phytochrome control.     

Pulsed radiolysis of perrhenate in neutral aqueous solution: Comparison with pertechnetate

Reaction of perrhenate with the aquated electron in neutral aqueous solution yields ReO₄^2? (k_f 1.3 × 10¹⁰ M^?1 s^?1), with an absorption maximum at 290 nm (ε 1700). This decays by a second-order path (k_d 1.5 × 10⁹ M^?1 s^?1) at a rate ～ 100-fold faster than the decay of TcO₄^2? under similar conditions.     

Primary Quantum Yield and Volume Change of Phytochrome-A Phototransformation Determined by Laser-Induced Optoacoustic Spectroscopy*

The primary quantum yield, Φ₁₇₀₀, for the photoconver-sion of the red-absorbing form of phytochrome, P_r, to the set of primary intermediates, Iⁱ₇₀₀, was redetermined by laser-induced optoacoustic spectroscopy at very low excitation laser fluences. The Iⁱ₇₀₀ value obtained is in the range of Φ_Pfr reported for the complete phototransformation P_r→ P_fr (J. M. Kelly and C. Lagarias, 1985, Biochemistry 24 , 4003–6010). An energy level of ca 150 kJ/ mol was found for the intermediates Iⁱ₇₀₀, i.e. ca 85% of the 0–0 level of P_r. Furthermore, a molecular expansion of 7 mL/mol (equivalent to 11 ų/molecule) was determined for the P_r→ Iⁱ₇₀₀ transformation. It reflects the protein reorganization induced by the geometrical pho-toisomerization of the chromophore, which results in changes of bonding interactions, in particular between the chromophore and its protein surrounding.