Formate Oxidation and Oxygen Reduction by Leaf Mitochondria '

Mitochondria isolated from the leaves of several plant species were investigated for the presence of NAD-linked formate dehydrogenase. The NADH produced was oxidized by the electron transport sequence and was coupled to ATP synthesis. The amounts of formate dehydrogenase, and, thereby, the capacity for formate-dependent 02 uptake, varied greatly among species. While no activity was detectable in mitochondria from soybean leaves, the rate of formate oxidation by spinach mitochondria was about one-half the rate of malate oxidation. In spinach, only mitochondria from green tissues oxidized formate. These last two observations raise questions as to the role of this reaction and the possible sources of the formate metabolized. The metabolism of formate in higher plants appears to proceed by three independent mechanisms. Formate is reduced to hydroxymethyl tetrahydrofolate, in which state it can participate in serine synthesis by the serine hydroxymethyl transferase reaction (16). Alternatively, formate can be oxidized to CO2 by two separate reactions. These are the peroxidatic action of catalase (7, 8) and the activity of NAD+-dependent formate dehydrogenase (7, 9). Both the serine hydroxymethyl transferase and the NAD-dependent formate dehydrogenase activities are mitochondrial, whereas the catalase reaction is peroxisomal (7, 15). The source of the formate metabolized by these reactions remains obscure. In higher plants, formate is often assumed to arise from the glyoxylate produced by photorespiration. Hydrogen peroxide, produced either by the glycolate oxidase reaction (16) or by isolated chloroplasts (17), can react nonenzymically with glyoxylate to form CO2 from the carboxyl carbon and formate from the methylene carbon. The extent to which this reaction occurs has been debated during recent years (5, 6, 11-13). Isolated soybean leaf cells metabolized added glycolate with approximately equal rates of CO2 loss by glycine decarboxylase activity and direct decarboxylation of glyoxylate (11). With these cells, however, CO2 is released from the methylene carbon of glycolate with different kinetics and inhibitor sensitivity than that from added formate (12). Sommerville et al. (13) have suggested that CO2 release directly from glyoxylate only occurs when insufficient amino donors preclude the transamination of glyoxylate to glycine. In this paper, mitochondrial formate oxidation by formate dehydrogenase was shown to be coupled to oxidative phosphorylation. The rate of oxidation varied substantially among mitochondria isolated from the leaves of different species and among preparations from different parts of the same plant. ' This work was supported by United States Department of Agriculture, Science and Education Administration, Competitive Research Grants Offlce Award 59-2161-0-1-490-0 and is Publication 80528 of the Idaho Agricultural Experiment Station. MATERIALS AND METHODS Tobacco (Nicotinia tabacum), lentils (Lens culinaris), and soybeans (Glycine max) were grown in a greenhouse. Spinach (Spinacia oleracea) was either greenhouse-grown or purchased locally. Peas (Pisum sativum), bush beans (Phaseolus vulgaris), chard (Beta vulgaris cicla), beets (Beta vulgaris), and lettuce (Lactuca sativa) were grown in a garden plot. Mitochondria were isolated by a modification of the method of Douce et al. (2). Leaf tissue was homogenized for 4 s in a Waring Blendor in 250 ml partially frozen slurry containing 0.3 M mannitol, 2 mm EDTA, 30 mm Hepes (pH 7.5), 0.2% (w/v) BSA, 0.6% (w/v) polyvinylpolypyrolidone, and 0.05% (v/v) 2-mercaptoethanol. Better coupling ratios were obtained if the BSA level was increased to 1% for bush beans and beets. The homogenate was rapidly filtered through four layers of cheesecloth and two layers of Miracloth (Chicopee Mills, Milltown, NJ) and centrifuged at 1,000g for 10 min. The supernatant was then centrifuged at l0,OOOg for 10 min. The pellet was resuspended in about 30 ml reaction mix, which was comprised of 0.3 M mannitol, 5 mm MgCl2, 10 mm KCI, 10 mm K-phosphate (pH 7.2), and 0.1% BSA (2). Resuspension was greatly facilitated by use of a No. 5 red sable artist brush. The mitochondrial preparation was centrifuged again at l,000g for 10 min, and the mitochondria were collected from the supernatant by centrifuging at 6,000g for 10 min (1). This pellet was either resuspended in the reaction mix and used directly or further purified by phase partitioning (4). Chloroplast protein contamination was corrected as described (2). 02 uptake was measured in a Clark-type 02 electrode (Hansatech Ltd., King's Lynn, Norfolk, England). The reaction volume was 1 ml. CO2 release was measured with [14C]formate, as described earlier (11, 12). Formate dehydrogenase activity was assayed by solubilizing 0.025 to 0.200 ml of the mitochondrial preparation in 2 ml 20 mM Hepes (pH 7.6) with 0.1% Triton X-100 and 1 mm NAD+. After the absorbance had stabilized, 15 mm Na Formate was added and the optical density increase monitored at 340 nm.