Role of the electrostatic loop charged residues in Cu, Zn superoxide dismutase

Abstract

We have expressed and characterized a mutant of Xenopus laevis Cu,Zn superoxide dismutase in which four highly conserved charged residues belonging to the electrostatic loop have been replaced by neutral side chains: Lysl20 + Leu, Asp130 + Gln, Glu131 + Gln, and Lys134 -+ Thr. At low ionic strength, the mutant enzyme is one of the fastest superoxide dismutases ever assayed (k = 6.7 X 10' M" s-' , at pH 7 and p = 0.02 M). Brownian dynamics simulations give rise to identical enzyme-substrate association rates for both wild-type and mutant enzymes, ruling out the possibility that enhancement of the activity is due to pure electrostatic factors. Comparative analysis of the experimental catalytic rate of the quadruple and single mutants reveals the nonadditivity of the mutation effects, indicating that the hyperefficiency of the mutant is due to a decrease of the energy barrier and/or to an alternative pathway for the diffusion of superoxide within the active site channel. At physiological ionic strength the catalytic rate of the mutant at neutral pH is similar to that of the wild-type enzyme as it is to the catalytic rate pH dependence. Moreover, mutation effects are additive. These results show that, at physiological salt conditions, electrostatic loop charged residues do not influence the diffusion pathway of the substrate and, if concomitantly neutralized, are not essential for high catalytic efficiency of the enzyme, pointing out the role of the metal cluster and of the invariant Arg 141 in determining the local electrostatic forces facilitating the diffusion of the substrate towards the active site.

Candida albicans SOD5 represents the prototype of an unprecedented class of Cu-only superoxide dismutases required for pathogen defense

“…It is also possible that the fungal family of Cu-only SODs evolved with an alternative electrostatic guidance system. In fact, an auxiliary electrostatic guidance system was previously proposed for SOD1, where the reaction rate of Xenopus laevis SOD1 was seen to increase rather than decrease when all of the electrostatic loop charges were neutralized by mutagenesis (45). In addition to substrate guidance, the extended electrostatic loop of SOD1 helps stabilize the zinc site, and the reverse is true: Zinc helps position the electrostatic loop (3,4,21).…”

Unique Features of the sodC-encoded Superoxide Dismutase from Mycobacterium tuberculosis, a Fully Functional Copper-containing Enzyme Lacking Zinc in the Active Site

“…Another highly conserved residue among both eukaryotic and prokaryotic enzymes is Asp 167 (Asp 122 in bovine SOD). Asp 167 is fundamental in the formation of the second coordination sphere, forming a bridge between copper and zinc ligands (His 44 and His 69 in bovine SOD, respectively), and is a key determinant of the active-site conformation and stability. This residue is conserved in M. tuberculosis, where it makes hydrogen bond contacts with the copper ligand His 84 and with the amide nitrogen of Phe 169 , but is far from the histidine residues corresponding to the ones coordinating zinc in the other SODs.…”

“…This is achieved in prokaryotes via a cluster of lysine residues in the S-S subloop and similarly in eukaryotic enzymes, where lysine residues in the electrostatic loop serve this purpose. At the physiological ionic strength, the metal cluster and the invariant Arg 199 (Arg 141 in bovine numbering) are mainly responsible for the enhancement of the substrate diffusion toward the active site (44). Although there are no charged residues either in the S-S subloop or in the dimerization loop (which corresponds to the eukaryotic electrostatic loop) in the mycobacterial SODs, the active site is surrounded by an area of positive potential centered on a lysine residue (Lys 198 in MtSOD) present in all mycobacterial SODs, which precedes the universally conserved Arg 199 and points toward the solvent (Figs.…”

Selective Inhibition of Fe- versus Cu/Zn-Superoxide Dismutases by 2,3-Dihydroxybenzoic Acid Derivatives.

“…Several studies, based on chemical modification and site-directed mutagenesis on charged residues of SODs, showed the existence of an electrostatic control of substrate diffusion involving positively charged residues. [18][19][20][21][22] It can be assumed that the catechol-induced loss of activity of E. coli Fe-SOD is attributable to covalent modifications occurring at these residues located at the entrance of the channel conducting towards the active metal ion (i.e. Lys 29, Lys 116 or Arg 170; Chart 2A).…”