Depolarized resonance light scattering by porphyrin and chlorophyll a aggregates.

A quantum mechanical model is developed for the observed resonance enhancement of light scattering by aggregates of electronically interacting chromophores. Aggregate size, monomer oscillator strength, extent of electronic coupling, and aggregate geometry are all important determinants of intensity in resonance light scattering (RLS) spectra. The theory also predicts the value of the depolarization ratio (rho(v)(90)) of RLS for a given aggregate geometry. These results are used to interpret the RLS depolarization ratios of four aggregates: tetrakis(4-sulfonatophenyl)porphine aggregated at low pH (rho(v)(90) = 0.17 at 488 nm), trans-bis(N-methylpyridinium-4-yl)-diphenylporphinato copper(II) aggregated in 0.2 M NaCl solution (rho(v)(90) = 0.13 at 450 nm) and on calf thymus DNA (rho(v)(90) = 0.20 at 454 nm), and chlorophyll a aggregates in formamide/water (rho(v)(90) = 0.23 and 0.32 at 469 and 699 nm, respectively). The analysis is consistent with a J-aggregate geometry for all four systems. Furthermore, the specific values of rho(v)(90) allow us to estimate the orientation of the monomer transition dipoles with respect to the long axis of the aggregate. We conclude that depolarized resonance light scattering spectroscopy is a powerful probe of the geometric and electronic structures of extended aggregates of strong chromophores.     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Both chlorophylls a and d are essential for the photochemistry in photosystem II of the cyanobacteria, Acaryochloris marina

We have measured the flash-induced absorbance difference spectrum attributed to the formation of the secondary radical pair, P(+)Q(-), between 270 nm and 1000 nm at 77 K in photosystem II of the chlorophyll d containing cyanobacterium, Acaryochloris marina. Despite the high level of chlorophyll d present, the flash-induced absorption difference spectrum of an approximately 2 ms decay component shows a number of features which are typical of the difference spectrum seen in oxygenic photosynthetic organisms containing no chlorophyll d. The spectral shape in the near-UV indicates that a plastoquinone is the secondary acceptor molecule (Q(A)). The strong C-550 change at 543 nm confirms previous reports that pheophytin a is the primary electron acceptor. The bleach at 435 nm and increase in absorption at 820 nm indicates that the positive charge is stabilized on a chlorophyll a molecule. In addition a strong electrochromic band shift, centred at 723 nm, has been observed. It is assigned to a shift of the Qy band of the neighbouring accessory chlorophyll d, Chl(D1). It seems highly likely that it accepts excitation energy from the chlorophyll d containing antenna. We therefore propose that primary charge separation is initiated from this chlorophyll d molecule and functions as the primary electron donor. Despite its lower excited state energy (0.1 V less), as compared to chlorophyll a, this chlorophyll d molecule is capable of driving the plastoquinone oxidoreductase activity of photosystem II. However, chlorophyll a is used to stabilize the positive charge and ultimately to drive water oxidation.     

CD Spectra of D1/D2/Cyt b559 Complax in Photodamaged Photosystem Ⅱ Reaction Center

The CD spectrum of photosystem Ⅱ reaction center D1/D2/Cyt b559 complex showed a strong reverse band with positive peak at 680 nm and negative peak at 660 nm in the red absorption region (Qy band). After the D1/D2/Cyt b559 complex was illuminated by strong light, the CD signals of the complex decreased significantly in the red region in which the negative peak still existed but the positive one disappeared. The result suggested that the CD signal of photosystem Ⅱ reaction center D1/D2/Cyt b559 complex not only came from the primary donor, P680, but also from other pigments such as from accessory Chl a or Pheo a.     

Site-directed mutations at D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803 modify the spectroscopic properties of the accessory chlorophyll in the D1-branch of the reaction center

D1-Thr179, which overlies the reaction center chlorophyll Chl D1 of Photosystem II was replaced with His and Glu through site-directed mutation in Synechocystis sp. PCC 6803. Spectroscopic characterization of the mutants indicates that, compared to wild type, the main bleaching in the triplet-minus-singlet absorbance difference spectrum and the electrochromic band shift in the (P680 (+)Q A (-)-P680Q A) absorbance difference spectrum are displaced to the red by approximately 2 nm in the D1-Thr179His mutant and to the blue by approximately 1 nm in the D1-Thr179Glu mutant. These difference spectra are compared with the absorbance difference spectra, measured on the same states in the D1-His198Gln mutant in which the axial ligand D1-His198 of the special pair chlorophyll, P D1, was replaced by glutamine. Together, these results give direct evidence that (a) the reaction center triplet state, produced upon charge recombination from (3)[P (+)Pheo (-)], is primarily localized on Chl D1; (b) the cation of the oxidized donor P (+) is predominantly localized on chlorophyll P D1 of the special pair; and (c) the Q Y band of the accessory chlorophyll Chl D1 is electrochromically shifted in response to charges on P (+) and Q A (-). Light-induced absorbance difference spectra (between 650 and 710 nm), associated with the oxidation of secondary donors and the reduction of Q A, exhibit a bleaching attributed to the oxidation of a Chl Z and strong electrochromic band shifts. On the basis of mutation-induced spectroscopic changes and of structure-based calculations, we conclude that the experimental spectra are best explained by a blue-shift of the Q Y band of the accessory chlorophyll Chl D1, arising from charges on Car D2 (+) and Chl ZD2 (+) and on reduced Q A.     

A spectroscopic and thermodynamic study of porphyrin/DNA supramolecular assemblies.

Assemblies of trans-bis(N-methylpyridinium-4-yl)diphenylporphine ions on the surface of calf thymus DNA have been studied using several spectroscopic techniques: absorbance, circular dichroism, and resonance light scattering. The aggregation equilibrium can be treated as a two-state system-monomer and assembly-each bound to the nucleic acid template. The aggregate absorption spectrum in the Soret region is resolved into two bands of Lorentzian line shape, while the DNA-bound monomer spectrum in this region is composed of two Gaussian bands. The Beer-Lambert law is obeyed by both porphyrin forms. The assembly is also characterized by an extremely large, bisignate induced circular dichroism (CD) profile and by enhanced resonance light scattering (RLS). Both the CD and RLS intensities depend linearly on aggregate concentration. The RLS result is consistent with a model for the aggregates as being either of a characteristic size or of a fixed distribution of sizes, independent of total porphyrin concentration or ionic strength. Above threshold values of concentration and ionic strength, the mass action expression for the equilibrium has a particularly simple form: K' = cac-1; where cac is defined as the "critical assembly concentration."offe dependence of the cac upon temperature and ionic strength (NaCl) has been investigated at a fixed DNA concentration. The value of the cac scales as the inverse square of the sodium chloride concentration and, from temperature dependence studies, the aggregation process is shown to be exothermic.     

Correction of spectra for studying dye aggregates by resonance light scattering

Resonance light scattering (RLS), a phenomenon of abrupt enhancement of Rayleigh light scattering in close proximity to an absorption band, is easily detectable in solutions of strongly absorbing chromophores, which form large aggregates with strong π-electronic coupling among the chromophores. RLS spectra need to be corrected for the sensitivity of the spectrofluorimeter as well as for the effects of internal light filter. A method for correcting the measured RLS is described. It was shown by the method that addition of KCl induces formation of extended supramolecular aggregates (probably of H-type) of the anionic dye merocyanine 540 in water. The RLS spectra of a photosensitizer m-tetra(hydroxyphenyl)chlorin (Foscan®) indicate formation of J-aggregates of this dye in aqueous medium.     

Interactions of Chlorophyll and Polypeptide Mixture with Bacterial Reaction Centres

In aqueous solutions of chlorophyll (Chl) a with synthesized polypeptides, at high ratios of Chl to polypeptides (about 75–150 µM to 500 µM) clusters of polypeptides and pigment molecules were formed. The main absorption maxima of more than one formed cluster were located at about 500 nm (Soret band) and in the region of 720–806 nm (red band). The formation of these clusters was fairly slow (some hours) at room temperature and even slower at 4 °C. The rate of cluster formation increased with the increase in Chl concentration. The addition of the even low amount of reaction centres (RCs), separated from the purple bacteria Rhodobacter sphaeroides, to the sample of Chl with polypeptides caused a very strong decrease in the efficiency of cluster formation, and a change in concentration ratios of various pigment-polypeptide aggregates. It was probably a competition between the interaction of Chl with polypeptides and with the RCs. The yield of thermal deactivation of the clusters was high, much higher than that for the RCs alone and it was different for various types of cluster. The clusters absorbing at 725–750 nm were fluorescent with maximum of emission at about 770 nm, whereas clusters absorbing at about 800 nm were nonfluorescent.     

Study of the interaction of hexa-amine cobalt (III) ion with DNA by a resonance light scattering technique and its analytical application.

The effect of hexa-amine cobalt cations on the DNA condensation in aqueous solution was investigated by resonance light scattering (RLS). When the relative concentration of hexa-amine cobalt (III) cations to DNA is in the appropriate range, the cations will induce DNA condensation and aggregation, which results in a strong RLS spectrum characterized by a peak at 290.0 nm. The RLS technique is a powerful tool for monitoring DNA condensation and, under optimal conditions, the enhanced RLS intensity at 290.0 nm was proportional to the concentration of DNA in the range 0.01-6.0 microg/mL. Based on this, a sensitive and convenient analysis method for the microdetermination of DNA was established. The detection limit (3 s) of calf thymus DNA by the proposed method is 1.9 ng/mL and few substances interfere in the DNA determination.     

Resonance light scattering determination of trace bisphenol A with signal amplification by aptamer–nanogold catalysis

HAuCl₄ was reduced by sodium citrate to prepare 10 nm gold nanoparticles (AuNPs) that were modified by the bisphenol A aptamer (Apt) to obtain an aptamer–nanogold probe (Apt‐AuNP) for bisphenol A (BPA). The probes were aggregated nonspecifically to form large clusters, which showed a strong resonance light scattering (RLS) peak at 520 nm, under preparation conditions (pH 7.6 Na₂HPO₄‐NaH₂PO₄ buffer and ultrasonication). Upon addition of BPA, the probe reacted specifically to form dispersed BPA‐Apt‐AuNP conjugates that exhibited strong catalysis of the two particle reactions of glucose‐Cu(II) and hydrazine hydrochloride‐Cu(II) with a strong RLS peak at 360 nm and 510 nm respectively. When the BPA concentration increased, the RLS intensity at 360 nm and 510 nm increased respectively. Accordingly, two new and highly‐sensitive RLS methods were established for the detection of BPA, using the Apt‐AuNP catalytic amplification. Copyright © 2013 John Wiley & Sons, Ltd.     

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.     

Linear dichroism of microalgae, developing thylakoids and isolated pigment-protein complexes in stretched poly(vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields. Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b(648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl a/b ratio of approx. 6 and the LD spectrum was positive with a maximum at 690-694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

Spectral analysis on origination of the bands at 437 nm and 475.5 nm of chlorophyll fluorescence excitation spectrum in Arabidopsis chloroplasts

Chlorophyll fluorescence has been often used as an intrinsic optical molecular probe to study photosynthesis. In this study, the origin of bands at 437 and 475.5 nm in the chlorophyll fluorescence excitation spectrum for emission at 685 nm in Arabidopsis chloroplasts was investigated using various optical analysis methods. The results revealed that this fluorescence excitation spectrum was related to the absorption characteristics of pigment molecules in PSII complexes. Moreover, the excitation band centred at 475.5 nm had a blue shift, but the excitation band at 437 nm changed relatively less due to induction of non‐photochemical quenching (NPQ). Furthermore, fluorescence emission spectra showed that this blue shift occurred when excitation energy transfer from both chlorophyll b (Chl b) and carotenoids (Cars) to chlorophyll a (Chl a) was blocked. These results demonstrate that the excitation band at 437 nm was mainly contributed by Chl a, while the excitation band at 475.5 nm was mainly contributed by Chl b and Cars. The chlorophyll fluorescence excitation spectrum, therefore, could serve as a useful tool to describe specific characteristics of light absorption and energy transfer between light‐harvesting pigments. Copyright © 2015 John Wiley & Sons, Ltd.     

A long-wave absorbing form of chlorophyll a responsible for the “red drop” in fluorescence at 298 °K and the F723 band at 77 °K

When Chlorella cells are ruptured at pH 4.6 by sonication in air, its absorption spectrum can be best explained if one assumes that a long-wave chlorophyll a form (Chl a 693) is preferentially destroyed. Using these preparations, and comparing them with the algal suspension and the sonicates prepared at pH 7.8 under argon, we make the following conclusions: (a) The red drop beginning at about 675–680 nm in the action spectrum^* of fluorescence at 298 °K must be due to the presence of a non-(or weakly) fluorescent form of chlorophyll a. We suggest that this form is Chl a 693. The red drop is absent in the aerobic sonicates. (b) The red drop in fluorescence in whole algal cells is not due to any errors in absorption measurements; this drop is clearly present in the anaerobic sonicates. (c) The emission band at 723 nm, discovered by in whole Chlorella cells at 77 °K, may be due to increased fluorescence efficiency of Chl a 693 at low temperature; the F723 band is absent in aerobic sonicates.     

Pigment-pigment interactions in PCP of Amphidinium carterae investigated by nonlinear polarization spectroscopy in the frequency domain

Peridinin-chlorophyll a-protein (PCP) is a unique antenna complex in dinoflagellates that employs peridinin (a carotenoid) as its main light-harvesting pigment. Strong excitonic interactions between peridinins, as well as between peridinins and chlorophylls (Chls) a, can be expected from the short intermolecular distances revealed by the crystal structure. Different experimental approaches of nonlinear polarization spectroscopy in the frequency domain (NLPF) were used to investigate the various interactions between pigments in PCP of Amphidinium carterae at room temperature. Lineshapes of NLPF spectra indicate strong excitonic interactions between the peridinin's optically allowed S(2) (1Bu(+)) states. A comprehensive subband analysis of the distinct NLPF spectral substructure in the peridinin region allows us to assign peridinin subbands to the two Chls a in PCP having different S(1)-state lifetimes. Peridinin subbands at 487, 501, and 535 nm were assigned to the longer-lived Chl, whereas a peridinin subband peaking at 515 nm was detected in both clusters. Certain peridinin(s), obviously corresponding to the subband centered at 487 nm, show(s) specific (possibly Coulombic?) interaction between the optically dark S(1)(2A(g)(-)) and/or intramolecular charge-transfer (ICT) state and S(1) of Chl a. The NLPF spectrum, hence, indicates that this peridinin state is approximately isoenergetic or slightly above S(1) of Chl a. A global subband analysis of absorption and NLPF spectra reveals that the Chl a Q(y)-band consists of two subbands (peaking at 669 and 675 nm and having different lifetimes), confirmed by NLPF spectra recorded at high pump intensities. At the highest applied pump intensities an additional band centered at S(1)/ICT transition of peridinin.     

A study of molecular interactions in light-harvesting complexes LHCIIb, CP29, CP26 and CP24 by Stark effect spectroscopy

Electric field-induced absorption changes (electrochromism or Stark effect) of the light-harvesting PSII pigment-protein complexes LHCIIb, CP29, CP26 and CP24 were investigated. The results indicate the lack of strong intermolecular interactions in the chlorophyll a (Chl a) pools of all complexes. Characteristic features occur in the electronic spectrum of Chl b, which reflect the increased values of dipole moment and polarizability differences between the ground and excited states of interacting pigment systems. The strong Stark signal recorded for LHCIIb at 650-655 nm is much weaker in CP29, where it is replaced by a unique Stark band at 639 nm. Electrochromism of Chl b in CP26 and CP24 is significantly weaker but increased electrochromic parameters were also noticed for the Chl b transition at 650 nm. The spectra in the blue region are dominated by xanthophylls. The differences in Stark spectra of Chl b are linked to differences in pigment content and organization in individual complexes and point to the possibility of electron exchange interactions between energetically similar and closely spaced Chl b molecules.     

Linear dichroism of microalgae,developing thylakoids and isolated pigment-protein complexes in stretched poly (vinyl alcohol) films at 77 K

A variety of unicellular algae, thylakoids from higher plants in different stages of maturity and isolated pigment-protein complexes were oriented in stretched polyvinyl alcohol films. Low temperature linear dichroism (LD) spectra of Chlorella pyrenoidosa and higher plant thylakoids in the films were very similar to those obtained after orientation of similar samples using magnetic or electric fields.Positive LD bands corresponding to Chl a (670) and (682) and negative bands due to Chl a (658) and Chl b (648) were resolved in spectra of the light harvesting Chl a/b protein. Chl b (648) and Chl a (658) and (670) were not seen in the LD spectrum of thylakoids from plants grown in intermittent light, the Chl b-less mutant of barley, Euglena gracilis or the cyanobacteria, Phormidium luridum and Anacystis nidulans, but did appear upon chloroplast maturation in Romaine lettuce and during the greening of etiolated and intermittent light plants. The highly oriented long wavelength Chl a (682) in the light-harvesting complex may represent residual PS II whose peak dichroism is centered at 681 nm. The PS I preparation had a Chl $\text{a}\text{b}$ ratio of approx. 6 and the LD spectrum was positive with a maximum at 690–694 nm and a band of lower amplitude at 652 nm. The minor LD band was not observed in PS I preparations from organisms that lack Chl b such as the cyanobacteria, intermittent light plants and the Chl b-less mutant of barley. We suggest that the 652 nm band is due to Chl b molecules associated with the antenna of PS I and are distinct from those on the light harvesting complex whose orientation is different. We also conclude that all the Chl a forms are oriented and that the long geometric axes of the pigment-protein complexes, as deduced from the configuration they assume in the stretched films, are axes that normally lie parallel to the plane of the native thylakoid.     

Singlet and triplet state transitions of carotenoids in the antenna complexes of higher-plant photosystem I

In this work, the spectroscopic characteristics of carotenoids associated with the antenna complexes of Photosystem I have been studied. Pigment composition, absorption spectra, and laser-induced triplet-minus-singlet (T-S) spectra were determined for native LHCI from the wild type (WT) and lut2 mutant from Arabidopsis thaliana as well as for reconstituted individual Lhca WT and mutated complexes. All WT complexes bind lutein and violaxanthin, while beta-carotene was found to be associated only with the native LHCI preparation and recombinant Lhca3. In the native complexes, the main lutein absorption bands are located at 492 and 510 nm. It is shown that violaxanthin is able to occupy all lutein binding sites, but its absorption is blue-shifted to 487 and 501 nm. The "red" lutein absorbing at 510 nm was found to be associated with Lhca3 and Lhca4 which also show a second carotenoid, peaking around 490 nm. Both these xanthophylls are involved in triplet quenching and show two T-S maxima: one at 507 nm (corresponding to the 490 nm singlet absorption) and the second at 525 nm (with absorption at 510 nm). The "blue"-absorbing xanthophyll is located in site L1 and can receive triplets from chlorophylls (Chl) 1012, 1011, and possibly 1013. The red-shifted spectral component is assigned to a lutein molecule located in the L2 site. A 510 nm lutein was also observed in the trimers of LHCII but was absent in the monomers. In the case of Lhca, the 510 nm band is present in both the monomeric and dimeric complexes. We suggest that the large red shift observed for this xanthophyll is due to interaction with the neighbor Chl 1015. In the native T-S spectrum, the contribution of carotenoids associated with Lhca2 is visible while the one of Lhca1 is not. This suggests that in the Lhca2-Lhca3 heterodimeric complex energy equilibration is not complete at least on a fast time scale.     

Spectroscopic studies of quinonoid species from pyridoxal 5'-phosphate

To establish the state of protonation of quinonoid species formed nonenzymically from pyridoxal phosphate (PLP) and diethyl aminomalonate, we have studied absorption spectra of the rapidly established steady-state mixture of species. We have evaluated the formation constant and the spectrum of the mixture of Schiff base and quinonoid species. For N-methyl-PLP a singly protonated species with a peak at 464 nm is formed from the unprotonated aldehyde and the conjugate acid of diethyl aminomalonate with a formation constant Kf of 240 M-1. The very intense absorption band with characteristic vibrational structure (most evident as a shoulder at 435 nm) is accompanied by a weaker, structured band at about 380 nm and a weak, broad band at 330 nm. We suggest that the 380-nm band may represent a tautomeric form of the quinonoid compound. Protonation of the phosphate group appears to affect the spectrum only slightly. The corresponding mixture of Schiff base and quinonoid species formed from PLP has a very similar spectrum at pH 6-7. It has a formation constant Kf of 230 M-1 and a pKa of 7.8, which must be attributed to the ring nitrogen atom. The dissociated species, which may be largely carbanionic, has a strong structured absorption band at 430 nm and a weaker one, again possibly a tautomer, in the 330-nm region. The analysis establishes that in all species a proton remains on either the phenolic oxygen or the imine nitrogen. Proton NMR spectroscopy, under some conditions, reveals only two components: free PLP and what appears to be Schiff base. However, we suggest that the latter may, in fact, be a quinonoid form, either alone or in rapid equilibrium with the Schiff base. Absorption spectra of quinonoid species formed in enzymes are analyzed and compared with the spectra of the nonenzymic species.