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The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54-60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 μmol g(-1) s(-1) protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20-100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with β-mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b 6 and an internal sequence of polyphenol oxidase.

We wanted to analyse the binding mode of small anion inhibitors to the zinc enzyme carbonic anhydrase in order to explore the binding of substrates and the catalytic mechanism of the enzyme. This was started by recording two data-sets by Laue diffraction to obtain the wanted structural information. In addition we wanted to test the capacity of the Laue method to show the small structural changes that are often associated with the catalytic activity of m any enzymes. To be able to exploit fully time-resolved crystallography the method should be able to detect such minor structural changes. The obtained Laue results did not agree with the expected molecular structures. Thus we needed to record monochromatic data-sets of the same states of the enzyme to confirm our results. All major findings from the Laue data agree with the monochromatic data. Stimulated by the unexpected findings we have continued the investigations of anion binding to carbonic anhydrase. We have studied both the zinc enzyme and replaced the native metal by cobalt which also yields an active enzyme. The accumulated picture of the ligand binding to the enzyme sheds new light on the substrate binding and on the catalytic mechanism.

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