Redox potential of chlorophyll d in vitro

Chlorophyll (Chl) d is a major chlorophyll in a novel oxygenic prokaryote Acaryochloris marina. Here we first report the redox potential of Chl d in vitro. The oxidation potential of Chl d was + 0.88 V vs. SHE in acetonitrile; the value was higher than that of Chl a (+ 0.81 V) and lower than that of Chl b (+ 0.94 V). The oxidation potential order, Chl b > Chl d > Chl a, can be explained by inductive effect of substituent groups on the conjugated π-electron system on the macrocycle. Corresponding pheophytins showed the same order; Phe b (+ 1.25 V) > Phe d (+ 1.21 V) > Phe a (+ 1.14 V), but the values were significantly higher than those of Chls, which are rationalized in terms of an electron density decrease in the π-system by the replacement of magnesium with more electronegative hydrogen. Consequently, oxidation potential of Chl a was found to be the lowest among Chls and Phes. The results will help us to broaden our views on photosystems in A. marina.     

Effects of molecular structures on reduction properties of formyl groups in chlorophylls and pheophytins prepared from oxygenic photosynthetic organisms

Reduction of the 7-formyl groups in chlorophyll (Chl) b and its demetalated compound pheophytin (Phe) b was kinetically analyzed by using tert-butylamine-borane complex (t-BuNH(2)·BH(3)), and was compared with that of the 3-formyl groups in Chl d and Phe d. Reduction kinetics of the 7-formyl group in Chl b was similar to that in Phe b in dichloromethane containing 5mM t-BuNH(2)·BH(3). Little difference of the reduction kinetics of the 7-formyl groups between Chl b and Phe b was in sharp contrast to the reduction kinetics of the 3-formyl groups in Chl d and Phe d: the 3-formyl group in Phe d was reduced 5.3-fold faster than that in Chl d. The 7-formyl groups in Chl b and Phe b were reduced more slowly than the 3-formyl groups in Chl d and Phe d, respectively. The difference of the reactivity between the 3- and 7-formyl groups was in line with (13)C NMR measurements of chlorophyllous pigments, in which the chemical shifts of carbon atoms in the 7-formyl groups of Chl b and Phe b were high-field shifted compared with those in the 3-formyl groups of Chl d and Phe d, respectively. These indicate that the 7-formyl groups in chlorophyllous pigments were less reactive for reduction to the corresponding hydroxymethyl groups than the 3-formyl groups due to the difference in electronic states of the formyl groups in the A- and B-rings of the chlorin macrocycle.     

Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.     

Function of chlorophyll d in reaction centers of photosystems I and II of the oxygenic photosynthesis of Acaryochloris marina

Reaction center chlorophylls (Chls) in photosystems II and I were studied in the isolated thylakoid membranes of a cyanobacterium, Acaryochloris marina, which contains Chls d and a as the major and minor pigments, respectively. The membranes contained PS I and II complexes at a 1.8:1 molar ratio on the basis of the spin densities on the tyrosine D radical and the photo-oxidized PS I primary donor (P740+). In the presence of ferricyanide, laser excitation induced bleach at 725 nm that recovered with time constants of 25 micros and 1.2 ms. The signal, designated P725, was suppressed by PS II inhibitors DCMU and hydroxylamine. The P725 spectrum was tentatively assigned to the absorption changes of the special pair Chl d, the accessory Chl d, and the acceptor pheophytin a in PS II. The addition of ascorbate induced the additional signal with a slow decay time constant of 4.5 ms. This signal showed a broad bleach at 740 nm and shift-type absorption changes at around 707 and 685 nm, which were assigned to the absorption changes of PS I special pair of Chl d (P740), the accessory Chl d, and the primary acceptor Chl a (A0), respectively. Mechanisms and the evolution of the Chl-d based reaction centers using far-red light are discussed together with the amino acid sequences of PS II D1 and D2 proteins.     

Chlorophyll b to chlorophyll a energy transfer kinetics in the CP29 antenna complex: a comparative femtosecond absorption study between native and reconstituted proteins

The energy transfer processes between Chls b and Chls a have been studied in the minor antenna complex CP29 by femtosecond transient absorption spectroscopy. Two samples were analyzed: the native CP29, purified from higher plants, and the recombinant one, reconstituted in vitro with the full pigment complement. The measurements indicate that the transfer kinetics in the two samples are virtually identical, confirming that the reconstituted CP29 has the same spectroscopic properties as the native one. In particular, three lifetimes (150 fs, 1.2 ps, and 5-6 ps) were identified for Chl b-652 nm to Chl a energy transfer and at least one for Chl b-640 nm (600-800 fs). Considering that the complexes bind two Chls b per polypeptide, the observation of more than two lifetimes for the Chl b to Chl a energy transfer, in both samples, clearly indicates the presence of the so-called mixed Chl binding sites--sites which are not selective for Chl a or Chl b, but can accommodate either species. The kinetic components and spectra are assigned to specific Chl binding sites in the complex, which provides further information on the structural organization.     

Stepwise two-photon excited fluorescence from higher excited states of chlorophylls in photosynthetic antenna complexes

Stepwise two-photon excited fluorescence (TPEF) spectra of the photosynthetic antenna complexes PCP, CP47, CP29, and light-harvesting complex II (LHC II) were measured. TPEF emitted from higher excited states of chlorophyll (Chl) a and b was elicited via consecutive absorption of two photons in the Chl a/b Qy range induced by tunable 100-fs laser pulses. Global analyses of the TPEF line shapes with a model function for monomeric Chl a in a proteinaceous environment allow distinction between contributions from monomeric Chls a and b, strongly excitonically coupled Chls a, and Chl a/b heterodimers/-oligomers. The analyses indicate that the longest wavelength-absorbing Chl species in the Qy region of LHC II is a Chl a homodimer with additional contributions from adjacent Chl b. Likewise, in CP47 a spectral form at approximately 680 nm (that is, however, not the red-most species) is also due to strongly coupled Chls a. In contrast to LHC II, the red-most Chl subband of CP29 is due to a monomeric Chl a. The two Chls b in CP29 exhibit marked differences: a Chl b absorbing at approximately 650 nm is not excitonically coupled to other Chls. Based on this finding, the refractive index of its microenvironment can be determined to be 1.48. The second Chl b in CP29 (absorbing at approximately 640 nm) is strongly coupled to Chl a. Implications of the findings with respect to excitation energy transfer pathways and rates are discussed. Moreover, the results will be related to most recent structural analyses.     

Metabolic engineering of the Chl d-dominated cyanobacterium Acaryochloris marina: production of a novel Chl species by the introduction of the chlorophyllide a oxygenase gene

In oxygenic photosynthetic organisms, the properties of photosynthetic reaction systems primarily depend on the Chl species used. Acquisition of new Chl species with unique optical properties may have enabled photosynthetic organisms to adapt to various light environments. The artificial production of a new Chl species in an existing photosynthetic organism by metabolic engineering provides a model system to investigate how an organism responds to a newly acquired pigment. In the current study, we established a transformation system for a Chl d-dominated cyanobacterium, Acaryochloris marina, for the first time. The expression vector (constructed from a broad-host-range plasmid) was introduced into A. marina by conjugal gene transfer. The introduction of a gene for chlorophyllide a oxygenase, which is responsible for Chl b biosynthesis, into A. marina resulted in a transformant that synthesized a novel Chl species instead of Chl b. The content of the novel Chl in the transformant was approximately 10% of the total Chl, but the level of Chl a, another Chl in A. marina, did not change. The chemical structure of the novel Chl was determined to be [7-formyl]-Chl d(P) by mass spectrometry and nuclear magnetic resonance spectroscopy. [7-Formyl]-Chl d(P) is hypothesized to be produced by the combined action of chlorophyllide a oxygenase and enzyme(s) involved in Chl d biosynthesis. These results demonstrate the flexibility of the Chl biosynthetic pathway for the production of novel Chl species, indicating that a new organism with a novel Chl might be discovered in the future.     

Spectral signatures of five hydroxymethyl chlorophyll a derivatives chemically derived from chlorophyll b or chlorophyll f

Photosynthesis Research - Chlorophylls (Chls) are pigments involved in light capture and light reactions in photosynthesis. Chl a, Chl b, Chl d, and Chl f are characterized by unique absorbance...     

Origin of the chlorophyll b formyl oxygen in Chlorella vulgaris.

Chlorophyll (Chl) b is an accessory light-harvesting pigment of plants and chlorophyte algae. Chl b differs from Chl a in that the 3-methyl group on ring B of chl a is replaced by a 3-formyl group on Chl b. The present study determined the biosynthetic origin of the Chl b formyl oxygen in in vivo labeling experiments. A mutant strain of the unicellular chlorophyte Chlorella vulgaris, which can not synthesize Chls when cultured in the dark but rapidly greens when transferred to the light, was grown in the dark for several generations to deplete Chls, and then the cells were transferred to the light and allowed to form Chls in a controlled atmosphere containing 18O2. Chl a and Chl b were purified from the cells and analyzed by high-resolution mass spectroscopy. Analysis of the mass spectra indicated that over 76% of the Chl a molecules had incorporated an atom of 18O. For Chl b, 58% of the molecules had incorporated an atom of 18O at one position and 34% of the molecules had incorporated an atom of 18O at a second position. These results demonstrate that the isocyclic ring keto oxygen of both Chl a and Chl b, as well as the formyl oxygen of Chl b, is derived from O2.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and beta-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H2O2 system. But they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Structural modeling of the Lhca4 Subunit of LHCI-730 peripheral antenna in photosystem I based on similarity with LHCII

Peripheral chlorophyll a/b binding antenna of photosystem I (LHCI) from green algae and higher plants binds specific low energy absorbing chlorophylls (red pigments) that give rise to a unique red-shifted emission. A three-dimensional structural model of the Lhca4 polypeptide from the LHCI from higher plants was constructed on the basis of comparative sequence analysis, secondary structure prediction, and homology modeling using LHCII as a template. The obtained model of Lhca4 helps to visualize protein ligands to nine chlorophylls (Chls) and three potential His residues to extra Chls. Central domain of the Lhca4 comprising the first (A) and the third (C) transmembrane (TM) helices that binds 6 Chl molecules and two carotenoids is conserved structurally, whereas the interface between the first and the second TM helices and the outer surface of the second TM helix differ significantly among the LHCI and LHCII polypeptides. The model of Lhca4 predicts a histidine residue in the second TM helix, a potential binding site for extra Chl in close proximity to Chls a5 and b5 (labeling by Kühlbrandt). The interpigment interactions in the formed pigment cluster are suggested to cause a red spectral shift in absorption and emission. Modeling of the LHCI-730 heterodimer based on the model structures of Lhca1 and Lhca4 allowed us to suggest potential sites of pigment-pigment interactions that might be formed upon heterodimerization or docking of the LHCI dimers to the surface of PSI.     

Chlorophyll and carotenoid degradation mediated by thylakoid-associated peroxidative activity in olives (Olea europaea) cv. hojiblanca

A peroxidative activity was found in solubilized thylakoid membranes of olives (Olea europaea) cv. hojiblanca that catalyses degradation of chloroplast pigments in the presence of H2O2 and 2,4-dichlorophenol (DCP). The intermediate products of this degradation were analyzed using HPLC with diode array detection and the results indicated that 13(2)-OH-chlorophyll a and 13(2)-OH-chlorophyll b were the primary catabolites. The peroxidative activity assosiated with the thylakoid membranes affected, not only chlorophyll a and chlorophyll b, but also other accessory pigments in the photosynthetic process, such as the carotenoids. Quantitatively, the progressive decrease of the ratios Chl a/b and total Chls a+b/carotenoids indicated a more rapid disappearance of Chl a than of Chl b and a faster degradation of Chls a+b than of carotenoids.     

Characterization of highly purified photosystem I complexes from the chlorophyll d-dominated cyanobacterium Acaryochloris marina MBIC 11017

Photochemically active photosystem (PS) I complexes were purified from the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina MBIC 11017, and several of their properties were characterized. PS I complexes consist of 11 subunits, including PsaK1 and PsaK2; a new small subunit was identified and named Psa27. The new subunit might replace the function of PsaI that is absent in A. marina. The amounts of pigments per one molecule of Chl d' were 97.0 +/- 11.0 Chl d, 1.9 +/- 0.5 Chl a, 25.2 +/- 2.4 alpha-carotene, and two phylloquinone molecules. The light-induced Fourier transform infrared difference spectroscopy and light-induced difference absorption spectra reconfirmed that the primary electron donor of PS I (P740) was the Chl d dimer. In addition to P740, the difference spectrum contained an additional band at 728 nm. The redox potentials of P740 were estimated to be 439 mV by spectroelectrochemistry; this value was comparable with the potential of P700 in other cyanobacteria and higher plants. This suggests that the overall energetics of the PS I reaction were adjusted to the electron acceptor side to utilize the lower light energy gained by P740. The distribution of charge in P740 was estimated by a density functional theory calculation, and a partial localization of charge was predicted to P1 Chl (special pair Chl on PsaA). Based on differences in the protein matrix and optical properties of P740, construction of the PS I core in A. marina was discussed.     

Universal chlorophyll equations for estimating chlorophylls <Emphasis Type="Italic">a,b, c</Emphasis>, and <Emphasis Type="Italic">d</Emphasis> and total chlorophylls in natural assemblages of photosynthetic organisms using acetone,methanol, or ethanol solvents

A universal set of equations for determining chlorophyll (Chl) a, accessory Chl b, c, and d, and total Chl have been developed for 90 % acetone, 100 % methanol, and ethanol solvents suitable for estimating Chl in extracts from natural assemblages of algae. The presence of phaeophytin (Ph) a not only interferes with estimates of Chl a but also with Chl b and c determinations. The universal algorithms can hence be misleading if used on natural collections containing large amounts of Ph. The methanol algorithms are severely affected by the presence of Ph and so are not recommended. The algorithms were tested on representative mixtures of Chls prepared from extracts of algae with known Chl composition. The limits of detection (and inherent error, ±95 % confidence limit) for all the Chl equations were less than 0.03 g m⁻³. The algorithms are both accurate and precise for Chl a and d but less accurate for Chl b and c. With caution the algorithms can be used to calculate a Chl profile of natural assemblages of algae. The relative error of measurements of Chls increases hyperbolically in diluted extracts. For safety reasons, efficient extraction of Chls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of Chls in natural assemblages of aquatic plants.     

Artificially produced [7-formyl]-chlorophyll d functions as an antenna pigment in the photosystem II isolated from the chlorophyllide a oxygenase-expressing Acaryochloris marina

Acaryochloris marina, a chlorophyll (Chl) d-dominated cyanobacterium, is a model organism for studying photosynthesis driven by far-red light using Chl d. Furthermore, studies on A. marina may provide insights into understanding how the oxygenic photosynthetic organisms adapt after the acquisition of new Chl. To solve the reaction mechanism of its unique photosynthesis, photosystem (PS) II complexes were isolated from A. marina and analyzed. However, the lack of a molecular genetic method for A. marina prevented us from conducting further studies. We recently developed a transformation system for A. marina and we introduced a chlorophyllide a oxygenase gene into A. marina. The resultant transformant accumulated [7-formyl]-Chl d, which has never been found in nature. In the current study, we isolated PS II complexes that contained [7-formyl]-Chl d. The pigment composition of the [7-formyl]-Chl d-containing PS II complexes was 1.96±0.04 Chl a, 53.21±1.00 Chl d, and 5.48±0.33 [7-formyl]-Chl d per two pheophytin a molecules. In contrast, the composition of the control PS II complexes was 2.01±0.06 Chl a and 62.96±2.49 Chl d. The steady-state fluorescence and excitation spectra of the PS II complexes revealed that energy transfer occurred from [7-formyl]-Chl d to the major Chl d species; however, the electron transfer was not affected by the presence of [7-formyl]-Chl d. These findings demonstrate that artificially produced [7-formyl]-Chl d molecules that are incorporated into PS II replace part of the Chl d molecules and function as the antenna. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Biofilm growth and near-infrared radiation-driven photosynthesis of the chlorophyll d-containing cyanobacterium Acaryochloris marina

The cyanobacterium Acaryochloris marina is the only known phototroph harboring chlorophyll (Chl) d. It is easy to cultivate it in a planktonic growth mode, and A. marina cultures have been subject to detailed biochemical and biophysical characterization. In natural situations, A. marina is mainly found associated with surfaces, but this growth mode has not been studied yet. Here, we show that the A. marina type strain MBIC11017 inoculated into alginate beads forms dense biofilm-like cell clusters, as in natural A. marina biofilms, characterized by strong O(2) concentration gradients that change with irradiance. Biofilm growth under both visible radiation (VIS, 400 to 700 nm) and near-infrared radiation (NIR, ～700 to 730 nm) yielded maximal cell-specific growth rates of 0.38 per day and 0.64 per day, respectively. The population doubling times were 1.09 and 1.82 days for NIR and visible light, respectively. The photosynthesis versus irradiance curves showed saturation at a photon irradiance of E(k) (saturating irradiance) >250 μmol photons m(-2) s(-1) for blue light but no clear saturation at 365 μmol photons m(-2) s(-1) for NIR. The maximal gross photosynthesis rates in the aggregates were ～1,272 μmol O(2) mg Chl d(-1) h(-1) (NIR) and ～1,128 μmol O(2) mg Chl d(-1) h(-1) (VIS). The photosynthetic efficiency (α) values were higher in NIR-irradiated cells [(268 ± 0.29) × 10(-6) m(2) mg Chl d(-1) (mean ± standard deviation)] than under blue light [(231 ± 0.22) × 10(-6) m(2) mg Chl d(-1)]. A. marina is well adapted to a biofilm growth mode under both visible and NIR irradiance and under O(2) conditions ranging from anoxia to hyperoxia, explaining its presence in natural niches with similar environmental conditions.     

The nature of the photosystem II reaction centre in the chlorophyll d-containing prokaryote, Acaryochloris marina.

Pigment-protein complexes enriched in photosystem II (PS II) have been isolated from the chlorophyll (Chl) d containing cyanobacterium, Acaryochloris marina. A small PS II-enriched particle, we call 'crude reaction centre', contained 20 Chl d, 0.5 Chl a and 1 redox active cytochrome b-559 per 2 pheophytin a, plus the D1 and D2 proteins. A larger PS II-enriched particle, we call 'core', additionally bound the antenna complexes, CP47 and CP43, and had a higher chlorophyll per pheophytin ratio. Pheophytin a could be photoreduced in the presence of a strong reductant, indicating that it is the primary electron acceptor in photosystem II of A. marina. A substoichiometric amount of Chl a (less than one chlorophyll a per 2 pheophytin a) strongly suggests that Chl a does not have an essential role in the photochemistry of PS II in this organism. We conclude that PS II, in A. marina, utilizes Chl d and not Chl a as primary electron donor and that the primary electron acceptor is one of two molecules of pheophytin a.     

Consistent Sets of Spectrophotometric Chlorophyll Equations for Acetone, Methanol and Ethanol Solvents

A set of equations for determining chlorophyll a (Chl a) and accessory chlorophylls b, c ₂ , c ₁ + c ₂ and the special case of Acaryochloris marina, which uses Chl d as its primary photosynthetic pigment and also has Chl a, have been developed for 90% acetone, methanol and ethanol solvents. These equations for different solvents give chlorophyll assays that are consistent with each other. No algorithms for Chl c compounds (c ₂ , c ₁ + c ₂) in the presence of Chl a have previously been published for methanol or ethanol. The limits of detection (and inherent error, ± 95% confidence limit), for chlorophylls in all organisms tested, was generally less than 0.1 μg/ml. The Chl a and b algorithms for green algae and land plants have very small inherent errors (< 0.01 μg/ml). Chl a and d algorithms for Acaryochloris marina are consistent with each other, giving estimates of Chl d/a ratios which are consistent with previously published estimates using HPLC and a rarely used algorithm originally published for diethyl ether in 1955. The statistical error structure of chlorophyll algorithms is discussed. The relative error of measurements of chlorophylls increases hyperbolically in diluted chlorophyll extracts because the inherent errors of the chlorophyll algorithms are constants independent of the magnitude of absorbance readings. For safety reasons, efficient extraction of chlorophylls and the convenience of being able to use polystyrene cuvettes, the algorithms for ethanol are recommended for routine assays of chlorophylls. The methanol algorithms would be convenient for assays associated with HPLC work.     

Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana

Chlorophyll (Chl) biosynthesis and degradation are the only biochemical processes on Earth that can be directly observed from satellites or other planets. The bulk of the Chls is found in the light-harvesting antenna complexes of photosynthetic organisms. Surprisingly little is known about the biosynthesis of Chl b, which is the second most abundant Chl pigment after Chl a. We describe here the expression and properties of the chlorophyllide a oxygenase gene (CAO) from Arabidopsis thaliana, which is apparently the key enzyme in Chl b biosynthesis. The recombinant enzyme produced in Escherichia coli catalyses an unusual two-step oxygenase reaction that is the 'missing link' in the chlorophyll cycle of higher plants.