Role of the nonheme Fe(II) center in the biosynthesis of the plant hormone ethylene.

The final step of ethylene biosynthesis in plants is catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). In addition to ACC, Fe(II), O2, CO2, and ascorbate are required for in vitro enzyme activity. Direct evidence for the role of the Fe(II) center in the recombinant avocado ACCO has now been obtained through formation of enzyme.(substrate or cofactor).NO complexes. These NO adducts convert the normally EPR-silent ACCO complexes into EPR-active species with structural properties similar to those of the corresponding O2 complexes. It is shown here that the ternary Fe(II)ACCO.ACC.NO complex is readily formed, but no Fe(II)ACCO.ascorbate.NO complex could be observed, suggesting that ascorbate and NO are mutually exclusive in the active site. The binding modes of ACC and the structural analog alanine specifically labeled with 15N or 17O were examined by using Q-band electron nuclear double resonance (ENDOR). The data indicate that these molecules bind directly to the iron through both the alpha-amino and alpha-carboxylate groups. These observations are inconsistent with the currently favored mechanism for ACCO, in which it is proposed that both ascorbate and O2 bind to the iron as a step in O2 activation. We propose a different mechanism in which the iron serves instead to simultaneously bind ACC and O2, thereby fixing their relative orientations and promoting electron transfer between them to initiate catalysis.

A new look at a time-worn system: oxidation of CuZn-SOD by H2O2.

This work summarizes observations from numerous investigators on the reaction of the copper-zinc superoxide dismutase with hydrogen peroxide at physiological pH in order to propose a likely sequence of events that leads to 2-oxo-histidine formation, copper loss, inactivation, and random and site-specific peptide fragmentation. New data is presented for the bovine liver enzyme that indicate copper is lost as the copper(I) form which immediately reacts with bathocuproine disulfonate to form the characteristic complex that absorbs at 485 nm. Studies in TRIS buffer ruled out the loss of copper(II) followed by reduction of the high potential copper(II)-bathocuproine disulfonate complex by buffer because TRIS is known not to reduce this complex. The rate of loss of copper(I) is not affected by the spin trap, 5,5'-dimethylpyrolline-N-oxide (DMPO), nor by replacing oxygen with argon in the reaction. In addition, changes in the native electrophoretic pattern that are correlated with copper loss and not peptide fragmentation are also unaffected by DMPO, argon, EDTA, or DTPA. These data are taken as indirect evidence that the formation of 2-oxo-histidine is the first oxidative event, unaffected by DMPO, that occurs at the bound oxidant and leads to loss of copper(I). Peptide fragmentation and the peroxidative activity of the dismutase are discussed in light of these observations.

Two applications using N,N'-diethyldithiocarbamate as a stain for copper in native polyacrylamide gels of superoxide dismutase.

N,N'-Diethyldithiocarbamate has been shown to be an analytical stain for copper in native polyacrylamide gels of the copper-zinc superoxide dismutase from purified preparations as well as from crude red cell extracts, i.e., lysates from which hemoglobin has been removed (Jewett, S. L., and Rocklin, A. M. (1994) Anal. Biochem. 217, 236-240). Applying this methodology, it was found that the relative amounts of copper-containing forms of copper-zinc superoxide dismutase (EC 1.15.1.1) from bovine red cell extracts did not change significantly with either the age or with hydrogen peroxide treatment of the red cells. Furthermore, no significant changes were seen in the specific activity of the dismutase in either type of experiment. These observations for both types of experiments are contrary to what was expected from similar studies reported in the literature. However, discrepancies may be accounted for by hemoglobin interference in indirect dismutase assays of the previous work. In the case of the peroxide treatment of red cells, however, there is an additional factor in that the dismutase is protected from peroxide-mediated changes in copper content and heterogeneity by the hemoglobin present. This protection was demonstrated in in vitro experiments using only a 24-fold excess of hemoglobin over the dismutase.

N,N'-diethyldithiocarbamate as a stain for copper-zinc superoxide dismutase in polyacrylamide gels of red cell extracts.

N,N'-Diethyldithiocarbamate reacts with copper in the copper-zinc superoxide dismutase (EC 1.15.1.1) in polyacrylamide gels to form stable yellow-brown bands that are quantifiable at 448 nm. This method of examining superoxide dismutase has been applied to crude extracts of the enzyme obtained from red cell lysates from which hemoglobin has been removed by chloroform-ethanol precipitation. This treatment did not affect the activity and heterogeneity of purified dismutase added to lysates and recovered by the same method. The bands that develop in the dithiocarbamate-stained gels of the extracts correspond exactly to the bands of dismutase activity obtained with a positive activity stain using dianisidine, indicating that the dismutase is the only copper protein that gives rise to these bands. The amount of superoxide dismutase in the bands, determined by comparing the areas under unknown peaks to areas obtained with a standard dismutase sample, agrees with the amount predicted from indirect superoxide dismutase activity measurements. Any color in gels due to trace hemoglobin or hemoglobin degradation products is bleached overnight during staining with the dithiocarbamate.

Variation of one unit of activity with oxidation rate of organic substrate in indirect superoxide dismutase assays.

Although numerous reviews and articles have been written about indirect superoxide dismutase assays, little has been reported about the variation of the amount of superoxide dismutase giving 50% inhibition (SOD50), defined as one unit of dismutase activity, with variations in assay conditions. This report indicates that there is a linear dependence of the SOD50 on the magnitude of the uninhibited rate of oxidation of the organic substance for two indirect assays, one involving epinephrine autoxidation and the other NADH oxidation. For one indirect assay involving an acceleration of the oxidation rate of an organic substrate, the amount of superoxide dismutase giving a doubling of the oxidation rate, defined as one unit of dismutase activity, also shows this linear dependence on the autoxidation rate of the organic substance. The significance of this dependence and its implications for experimenters using these assays is discussed.