Activation of the Proton Transfer Pathway in Catalysis by Iron Superoxide Dismutase

Abstract

Catalysis by Escherichia coli and Porphyromonas gingivalis iron superoxide dismutase was activated by addition of primary amines, as measured by pulse radiolysis and stopped-flow spectrophotometry. This activation was saturable for most amines investigated, and a free energy plot of the apparent second-order rate constant of activation was linear as a function of the pK a of the amine, indicating activation by proton transfer. Amines provide an alternate rather than the only pathway for proton transfer, and catalysis was appreciable in the absence of amines. Solvent hydrogen isotope effects were near unity for amine activation, which is consistent with rate-contributing proton transfer if the pK a of the proton acceptor on the enzyme is not in the region of the pK a values of the amines studied, from 7.8 to 10.6. The activation of catalysis by these amines was uncompetitive with respect to superoxide, interpreted as proton transfer in a ternary complex of amine with the enzyme-bound peroxide dianion.The superoxide dismutases (SODs) 1 are cyclic redox enzymes that provide protection against oxidative damage caused by the reactions of the superoxide radical anion O 2 . . SODs have been characterized with manganese, iron, copper/zinc, or nickel as the redox active metal (1-3). The catalysis of the dismutation of superoxide occurs in two stages in which the metal ion, designated M in Reactions 1 and 2, alternates between the M 3ϩ and M 2ϩ forms (2-4),Iron superoxide dismutase (Fe-SOD), found in prokaryotes, and Mn-SOD, found in mitochondria as well as in prokaryotes, are highly similar in sequence (5) and in crystal structure (6, 7). Despite this high degree of structural homology, substitution of one metal for the other generally results in an inactive enzyme (8,9). A subclass of SOD exists that is able to utilize either manganese or iron as catalytic metal ion; these are termed cambialistic SODs and are of significant interest from the perspective of metal specificity as well as catalytic mechanism. A well studied cambialistic SOD is that from Porphyromonas gingivalis, the crystal structure of which is reported for the manganese-and iron-containing forms (10). Although they all share common features of their catalytic pathways, the copper/ zinc-and nickel-containing SODs have structures unrelated to the manganese-and iron-containing SODs (2, 11).The crystal structures of the iron-and manganese-containing superoxide dismutases generally show a trigonal bipyramidal geometry about the oxidized metal with three histidines, one aspartic acid, and one solvent molecule as ligands (6, 7). Major features of the active site appear very similar in each enzyme, including the highly conserved hydrogen-bonded network connecting the metal-coordinated solvent molecule and residues Gln-69 2 (in Fe-SOD) or Gln-143 (in Mn-SOD), Tyr-34, His-30, and Tyr-166 from the adjacent subunit. Although this active site Gln emanates from a different backbone position in Fe-SOD and Mn-SOD, the side chain orientation is very similar in both. Thi...

Superoxide Dismutases and Superoxide Reductases

“…Thus, the H 2 O ligand is believed to serve as a local proton donor to nascent peroxide, 132b , 133 with the active site hydrogen-bond network providing a proton relay 134 that mediates proton movement, determines the placement of protons, 133a and enables the active site to draw upon bulk water for the second proton, 135 which is required for product release (eq 17 ). 131 , 136 As is shown in Scheme 3 , the catalytic mechanism can be broken down into five steps that will be discussed individually below.…”

Transition metal complexes bearing flexible N3 or N3O donor ligands: Reactivity toward superoxide radical anion and hydrogen peroxide

“…electrostatic repulsion when anionic ligand is present vs. the lack of it) cannot be elucidated clearly, because (as the structural comparison of 2-Cl and 2-Me reveals) the carboxylate site is occupied by a chloride when methylcarboxyl group is present. Since a detailed summary of literature data was provided in our recent publications [26,27], we only wish to note here that these values are comparable to those of the more effective scavengers, but remain much below the k cat of the native enzymes that are all very close or in excess of 10 9 M −1 s −1 [46,[48][49][50][51].…”

Improved synthesis and purification of methylated amanitins using diazomethane*

“…The conversion of a-amanitin (AMA) to 6'-O-methyl-cr-amanitin (meAMA) using diazomethane was described as an intermediate in the synthesis of labeled amatoxins by Wieland & Fahrmeir (8). This derivative provided a reactant for the controlled periodate oxidation of the y,F-dihydroxyisoleucine moiety.…”