Cobalt Metabolism in Plants

Interactions between cobalt and several essential enzymes have been demonstrated in plants and animals. Two metal-bound intermediates formed by CO₂⁺ activate ribulose-1,5 bisphosphate carboxylase/ oxygenase (EC 4.1.1.39). Studies by electron paramagnetic resonance (EPR) spectroscopy have shown the activity to be dependent on the concentration of ribulose 1,5 bisphosphate (23). This finding suggested that the enzyme-metal coordinated ribulose 1,5 bisphosphate and an enzyme-metal coordinated enediolate anion of it, where bound ribulose 1,5 bisphosphate appears first, constitute the two EPR detectable intermediates, respectively.

Ganson and Jensen (56) showed that the prime molecular target of glyphosate (N-[phosphonomethyl] glycine), a potent herbicide and antimicrobial agent, is known to be the shikimatepathway enzyme 5-enol-pyruvylshikimate-3-phosphate synthetase. Inhibition by glyphosate of an earlier pathway enzyme that is located in the cytosol of higher plants, 3-deoxy-D-arabinoheptulosonate- 7 phosphate synthase (DS-Co), has raised the possibility of dual enzyme targets in vivo. Since the observation that magnesium or manganese can replace cobalt as the divalentmetal activator of DS-Co, it has now been possible to show that the sensitivity of DS-Co to inhibition by glyphosate is obligately dependent on the presence of cobalt. Evidence for a cobalt(II):glyphosate complex with octahedral coordination was obtained through examination of the effect of glyphosate on the visible electronic spectrum of aqueous solutions of CoCl₂.

Two inhibition targets of cobalt and nickel were studied on oxidation–reduction enzymes of spinach (Spinacia oleracea L.) thylakoids. Compounds of complex ions and coordination compounds of cobalt and chromium were synthesized and characterized (57). Their chemical structures and the oxidation states of their metal centers remained unchanged in solution. Neither chromium(III) chloride (CrCl₃) nor hexamminecobalt(III) chloride [Co(NH₃)₆C13] inhibited photosynthesis. Some other coordination compounds inhibited ATP synthesis and electron flow (basal phosphorylating, and uncoupled) behaving as Hill-reaction inhibitors, with the compounds targeting electron transport from photosystem II (P680 to plastoquinones, QA and QB, and cytochrome). The final step in hydrocarbon biosynthesis involves the loss of cobalt from a fatty aldehyde (58). This decarbonylation is catalyzed by microsomes from Botyrococcus braunii. The purified enzyme releases nearly one mole of cobalt for each mole of hydrocarbon. Electron microprobe analysis revealed that the enzyme contains cobalt. Purification of the decarbonylase from B. braunii grown in ⁵⁷CoCl₂ showed that ⁵⁷Co co-eluted with the decarbonylase. These results indicate that the enzyme contains cobalt that might be part of a Co-porphyrin, although a corrin structure (as in vitamin B₁₂) cannot be ruled out. These results strongly suggest that biosynthesis of hydrocarbons is effected by a microsomal Co-porphyrin-containing enzyme that catalyzes decarbonylation of aldehydes and, thus, reveals a biological function for cobalt in plants (58).

The role of hydrogen bonding in soybean (Glycine max Merr.) leghemoglobin was studied (59,60). Two spectroscopically distinct forms of oxycobaltous soybean leghemoglobin (oxyCoLb), acid and neutral, were identified by electron spin echo envelope modulation. In the acid form, a coupling to 2H was noted, indicating the presence of a hydrogen bond to bound oxygen. No coupled 2H occurred in the neutral form (60). The oxidation–reduction enzymes of spinach thylakoids are also affected by chromium and cobalt (23,57).

The copper chaperone for the superoxide dismutase (CCS) gene encodes a protein that is believed to deliver copper to Cu–Zn superoxide dismutase (CuZnSOD). The CCS proteins from different organisms share high sequence homology and consist of three distinct domains, a CuZnSOD-like central domain flanked by two domains, which contain putative metal-binding motifs. The CO₂⁺-binding properties of proteins from arabidopsis and tomato (Lycopersicon esculentum Mill.) were characterized by UV–visible and circular dichroism spectroscopies and were shown to bind one or two cobalt ions depending on the type of protein. The cobalt-binding site that was common in both proteins displayed spectroscopic characteristics of CO₂⁺ bound to cysteine ligands (61).

The inhibition of photoreduction reactions by exogenous manganese chloride (MnCl₂) in Tristreated photosystem II (PSII) membrane fragments has been used to probe for amino acids on the PSII reaction-center proteins, including the ones that provide ligands for binding manganese (62,63). Inhibition of photooxidation may involve two different types of high-affinity, manganesebinding components: (a) one that is specific for manganese, and (b) others that bind manganese, but may also bind additional divalent cations such as zinc and cobalt that are not photooxidized by PSII. Roles for cobalt or zinc in PSII have not been proposed, however.