Nitric oxide (NO) is a widespread signaling molecule, and numerous targets of its action exist in plants. Whereas the activity of NO in erythrocytes, microorganisms, and invertebrates has been shown to be regulated by several hemoglobins, the function of plant hemoglobins in NO detoxification has not yet been elucidated. Here, we show that Arabidopsis thaliana nonsymbiotic hemoglobin AHb1 scavenges NO through production of S-nitrosohemoglobin and reduces NO emission under hypoxic stress, indicating its role in NO detoxification. However, AHb1 does not affect NO-mediated hypersensitive cell death in response to avirulent Pseudomonas syringae, suggesting that it is not involved in the removal of NO bursts originated from acute responses when NO mediates crucial defense signaling functions.

The biochemical properties of PsbS protein, a nuclearencoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyllbinding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca 2؉ -binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [ 14 C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH. Photosystem II (PSII)1 of higher plants is a multisubunit membrane complex composed of many polypeptides that are encoded by the chloroplast or nuclear genes. Chloroplast gene products are located in the core complex where electron transport reactions are catalyzed, whereas the surrounding lightharvesting system is composed of nuclear-encoded chlorophyll a/b/xanthophyll proteins belonging to the Lhc family (1). Lhc proteins of PSII include Lhcb1-3 gene products that form heterotrimeric complexes (LHCII) located peripherally in the PSII⅐LHCII supercomplex and Lhcb4 -6 proteins, which form a layer of monomeric subunits located between the core complex and trimeric LHCII. Besides the role of harvesting light and transferring excitation energy to the PSII reaction center (RC), Lhc proteins are involved in regulative mechanisms aimed at both the optimization of excitation energy distribution between PSI and PSII and the protection of the PSII RC from photoinhibition when absorbed light exceeds the electron transport capacity of the chloroplast. The former mechanisms acting at moderate to low light intensity include the reversible phosphorylation of LHCII, which leads to the detachment of phospho-LHCII from PSII and its migration to stroma membranes where it transfers energy to PSI (2). The mechanisms of protection from photoinhibition are elicited at high light intensity and include the reversible phosphorylation of Lhcb4 (CP29) (3) and the xanthophyll cycle-dependent non-photochemical energy quenching (NPQ). In excess light conditions...

The nonsymbiotic hemoglobins, AHb1 and AHb2, have recently been isolated from Arabidopsis thaliana. Using steady-state and time-resolved spectroscopic methods, we show that Fe2+ AHb1 contains a mixture of penta- and hexacoordinated heme, while Fe2+ AHb2 is fully hexacoordinated. In the CO complexes, polar interactions and H-bonds with the ligand are stronger for AHb1 than for AHb2. The ligand binding kinetics are substantially different, reflecting the distribution between the penta- and hexacoordinated species, and indicate that protein dynamics and ligand migration pathways are very specific for each of the two proteins. In particular, a very small, non-exponential geminate rebinding observed in AHb1 suggests that the distal heme cavity is connected with the exterior by a relatively open channel. The large, temperature-dependent geminate rebinding observed for AHb2 implies a major role of protein dynamics in the ligand migration from the distal cavity to the solvent. The structures of AHb1 and AHb2, modeled on the basis of the homologous rice hemoglobin, exhibit a different cavity system that is fully compatible with the observed ligand binding kinetics. Overall, these kinetic and structural data are consistent with the putative NO-dioxygenase activity previously attributed to AHb1, whereas the role of AHb2 remains elusive.

DOPA decarboxylase (DDC) is responsible for the synthesis of the key neurotransmitters dopamine and serotonin via decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan, respectively. DDC has been implicated in a number of clinic disorders, including Parkinson's disease and hypertension. Peripheral inhibitors of DDC are currently used to treat these diseases. We present the crystal structures of ligand-free DDC and its complex with the anti-Parkinson drug carbiDOPA. The inhibitor is bound to the enzyme by forming a hydrazone linkage with the cofactor, and its catechol ring is deeply buried in the active site cleft. The structures provide the molecular basis for the development of new inhibitors of DDC with better pharmacological characteristics.

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Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with Ki and Kinact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with [2-14C]5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.

Dopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa into 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha-methyldopamine, which does not accumulate during catalysis. The ketone has been identified by high-performance liquid chromatography and mass spectroscopic analysis, and ammonia by means of glutamate dehydrogenase. Molecular oxygen is consumed during the reaction in a 1:2 molar ratio with respect to the products. The kcat and Km of this reaction were determined to be 5.68 min-1 and 45 microM, respectively. When the reaction is carried out under anaerobic conditions, alpha-methyldopamine is formed in a time-dependent manner and neither ammonia nor ketone is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with kinact of 0. 012 min-1 and Ki of 39.3 microM. Free 3,4-dihydroxyphenylacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a kinact/Ki value similar to that of alpha-methylDopa. d-Dopa, a competitive inhibitor of DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the ketone generated by the reaction of DDC with alpha-methylDopa dissociates from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by NaB3H4 reduction and chymotryptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modification. Together with the characterization of the adduct released from the inactivated DDC, these data suggest that the enzyme is inactivated by trapping the coenzyme in a ternary adduct with ketone and the active site lysine. As recently reported for serotonin (5-HT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem. 271, 23954-23959], the conversion of dopamine (DA) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC is accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3, 4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of these two molecules and DDC-bound PLP.

Cystathionine β-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T. gondii CGL gene encodes a functional enzyme. Herein, we cloned and biochemically characterized cDNA encoding CBS from T. gondii (TgCBS), which represents a first example of protozoan CBS that does not bind heme but possesses two C-terminal CBS domains. We demonstrated that TgCBS can use both serine and O-acetylserine to produce cystathionine, converting these substrates to an aminoacrylate intermediate as part of a PLP-catalyzed β-replacement reaction. Besides a role in cysteine biosynthesis, TgCBS can also efficiently produce hydrogen sulfide, preferentially via condensation of cysteine and homocysteine. Unlike the human counterpart and similar to CBS enzymes from lower organisms, the TgCBS activity is not stimulated by S-adenosylmethionine. This study establishes the presence of an intact functional reverse transsulfuration pathway in T. gondii and demonstrates the crucial role of TgCBS in biogenesis of H 2 S.