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Biochemical aspects of the Xanthophyll cycle

Arvidsson, Per-Ola (1996)
Abstract
Photosynthesis is the process through which the energy from the sun is converted into chemically bound energy stored in carbohydrates. Due to large variations in light intensities and stress situations, plants must be able to regulate the absorbed energy since excess energy would otherwise damage the plant. One of several photoprotective mechanisms involves the xanthophyll cycle. A copper binding protein was isolated from spinach photosystem II particles. The protein was identified, by partial amino acid sequencing, as CP26. EPR data indicated the presence of nitrogen and oxygen ligands, but no sulfur. We suggest that the copper-containing CP26 is identical to the enzyme zeaxanthin epoxidase, which converts zeaxanthin to violaxanthin. The... (More)
Photosynthesis is the process through which the energy from the sun is converted into chemically bound energy stored in carbohydrates. Due to large variations in light intensities and stress situations, plants must be able to regulate the absorbed energy since excess energy would otherwise damage the plant. One of several photoprotective mechanisms involves the xanthophyll cycle. A copper binding protein was isolated from spinach photosystem II particles. The protein was identified, by partial amino acid sequencing, as CP26. EPR data indicated the presence of nitrogen and oxygen ligands, but no sulfur. We suggest that the copper-containing CP26 is identical to the enzyme zeaxanthin epoxidase, which converts zeaxanthin to violaxanthin. The other enzyme involved in the xanthophyll cycle, the violaxanthin de-epoxidase, converts violaxanthin to zeaxanthin via antheraxanthin. Formation of zeaxanthin is believed to protect the plant from excess light by dissipating excess energy as heat. This enzyme was purified more than 5000-fold by gel filtration, hydrophobic interaction chromatography (HIC), and DEAE anion exchange chromatography. The enzyme was identified as a 43 kDa protein by SDS-PAGE and gel filtration. The isolated violaxanthin de-epoxidase shows a unique N-terminal sequence and we suggest that the enzyme is encoded by nuclear DNA. The enzyme was found to have a Km for ascorbate that is dependent on pH and that the acid form of ascorbate is the substrate for the enzyme. The release of the violaxanthin de-epoxidase from the thylakoids using sonication was found to be strongly pH-dependent with a cooperativity of 4 with respect to protons. We found that addition of purified violaxanthin de-epoxidase to thylakoid membranes could convert violaxanthin to zeaxanthin from both sides of the thylakoid membrane. We therefore conclude that this conversion takes place in the lipid matrix and not in the pigment protein complexes. We also found that the availability of violaxanthin is strongly temperature-dependent and conclude that temperature influences the partition of violaxanthin between the pigment protein complexes and the lipid matrix. A new and novel irreversible inhibition method for violaxanthin de-epoxidase was developed involving the reducing agent DTT and the SH-reagent iodoacetamide. From the pH dependence of this inhibition, we suggest that violaxanthin de-epoxidase undergoes a conformational change upon membrane binding. (Less)
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author
opponent
  • Prof Dr Krause, Henrich
publishing date
type
Thesis
publication status
published
subject
keywords
copper, zeaxanthin epoxidase, violaxanthin de-epoxidase, purification, photoprotection, pH-dependence, ascorbate, xanthophyll cycle, Plant biochemistry, Växtbiokemi
pages
47 pages
defense location
Sölvegatan 35 Plant Biochemistry
defense date
1996-04-26 10:15
external identifiers
  • Other:ISRN: LUNKDL/NKVK--96/1008--SE
ISBN
91-628-2010-9
language
English
LU publication?
no
id
644d41a2-b493-497c-a399-33ccfd4da716 (old id 28375)
date added to LUP
2007-06-11 13:40:15
date last changed
2016-09-19 08:45:17
@misc{644d41a2-b493-497c-a399-33ccfd4da716,
  abstract     = {Photosynthesis is the process through which the energy from the sun is converted into chemically bound energy stored in carbohydrates. Due to large variations in light intensities and stress situations, plants must be able to regulate the absorbed energy since excess energy would otherwise damage the plant. One of several photoprotective mechanisms involves the xanthophyll cycle. A copper binding protein was isolated from spinach photosystem II particles. The protein was identified, by partial amino acid sequencing, as CP26. EPR data indicated the presence of nitrogen and oxygen ligands, but no sulfur. We suggest that the copper-containing CP26 is identical to the enzyme zeaxanthin epoxidase, which converts zeaxanthin to violaxanthin. The other enzyme involved in the xanthophyll cycle, the violaxanthin de-epoxidase, converts violaxanthin to zeaxanthin via antheraxanthin. Formation of zeaxanthin is believed to protect the plant from excess light by dissipating excess energy as heat. This enzyme was purified more than 5000-fold by gel filtration, hydrophobic interaction chromatography (HIC), and DEAE anion exchange chromatography. The enzyme was identified as a 43 kDa protein by SDS-PAGE and gel filtration. The isolated violaxanthin de-epoxidase shows a unique N-terminal sequence and we suggest that the enzyme is encoded by nuclear DNA. The enzyme was found to have a Km for ascorbate that is dependent on pH and that the acid form of ascorbate is the substrate for the enzyme. The release of the violaxanthin de-epoxidase from the thylakoids using sonication was found to be strongly pH-dependent with a cooperativity of 4 with respect to protons. We found that addition of purified violaxanthin de-epoxidase to thylakoid membranes could convert violaxanthin to zeaxanthin from both sides of the thylakoid membrane. We therefore conclude that this conversion takes place in the lipid matrix and not in the pigment protein complexes. We also found that the availability of violaxanthin is strongly temperature-dependent and conclude that temperature influences the partition of violaxanthin between the pigment protein complexes and the lipid matrix. A new and novel irreversible inhibition method for violaxanthin de-epoxidase was developed involving the reducing agent DTT and the SH-reagent iodoacetamide. From the pH dependence of this inhibition, we suggest that violaxanthin de-epoxidase undergoes a conformational change upon membrane binding.},
  author       = {Arvidsson, Per-Ola},
  isbn         = {91-628-2010-9},
  keyword      = {copper,zeaxanthin epoxidase,violaxanthin de-epoxidase,purification,photoprotection,pH-dependence,ascorbate,xanthophyll cycle,Plant biochemistry,Växtbiokemi},
  language     = {eng},
  pages        = {47},
  title        = {Biochemical aspects of the Xanthophyll cycle},
  year         = {1996},
}