EPR studies of ribulose-1,5-bisphosphate carboxylase/oxygenase activated With Cu^2^+ - Styring, S.; Braanden, R.; Nilsson, T. (Elsevier)
Ribulose-1, 5-bisphosphate (RuBP) carboxylase/oxygenase catalyzes the primary reaction in two metabolic pathways in plants. A:in CO"2fixation: RuBP = CO"2 -〉 2,3-phosphoglyceric acid (PGA) B: In photorespiration: RuBP = O"2 -〉 PGA + phosphoglycolateIn the carboxylase reaction a 6-carbon intermediate has been identified. In the oxygenase reaction a 5-carbon peroxointermediate has been suggested. However, there is little experimental evidence for its existence [1].RuBP carboxylase/oxygenase requires a divalent metal ion for activity. The metal ion stabilizes the carbamate formed between the enzyme and a CO"2 molecule in the activation process [2]. Whether the metal also has a function during catalysis is not clear. Mg^2^+ is the normal activator in vivo but the enzyme is also activated by Fe^2^+, Mn^2^+, Co^2^+ and Ni^2^+.Cu^2^+ gives little activity and has not been much studied in carboxylase research. Despite this, Cu^2^+ may be a useful paramagnetic probe.Our results show that Cu^2^+ in the presence of HCO^-"3 (50 mM) is specifically bound to the enzyme. The high field part of the EPR spectrum shows nitrogen hyperfine splitting indicating that the metal in the Enz-CO"2Cu^2^+ complex has at least one nitrogen ligand [3]. The yield of the specific EPR signal is lowered by Mg^2^+ indicating that Cu^2^+ binds in or very near the normal metal activator site [3].When excess substrate (10 mM RuBP) is added to the Cu^2^+/Co"2 activated enzyme another EPR spectrum is obtained (Fig. 1). This signal has very narrow hyperfine lines which is only compatible with oxygen atoms being liganded to the Cu^2^+ ion. The nitrogen ligand in the EnzCO"2Cu^2^+-complex must therefore have been displaced by an oxygen atom probably derived from RuBP. This experiment has two important implications. First it proves that Cu^2^+ binds in the active site of the enzyme and secondly it implies that the metal is involved in substrate binding to the active site [3].When stoichiometric amounts of RuBP (0.5 mM) are added to the Cu^2^+/CO"2 activated enzyme a transient EPR signal is obtained. This signal is converted to a third signal with time. Both these signal possess the same narrow hyperfine lines as the EnzCO"2Cu^2^+-RuBP complex (Fig. 1) but have different parameters. The third signal can also be obtained by addition of 3-PGA to the EnzCO"2Cu^2^+ complex [4].The transient signal is dependent on the concentration of oxygen. In the presence of ^1^7O"2 the signal is broadened. This proves that at least one oxygen ligand to Cu^2^+ is derived from O"2. Preliminary experiments have shown that this effect is not derived from ^1^7O incorporated in any of the expected products of the oxygenase reaction. H"2O"2 and O"2 are also excluded as ligands. We therefore suggest that the transient EPR signal is derived from a peroxointermediate formed in the oxygenase reaction. If so the Cu^2^+-activated enzyme is very attractive for further research directed towards an understanding of the oxygenase reaction.
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