Disulfide - mutant SOD 1 is active and has metal binding like wild type 1 Insights into the Role of the Unusual Disulfide Bond in Copper - Zinc Superoxide

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human and yeast SOD1 (hSOD and ySOD) lacking the disulfide bond. We determined X-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. Addition of copper to this four-zinc protein gave properly reconstituted 2Cu2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild-type hSOD1. In contrast, addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found they were enzymatically active at approximately 10% of the wild-type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic http://www.jbc.org/cgi/doi/10.1074/jbc.M114.588798 The latest version is at JBC Papers in Press. Published on December 1, 2014 as Manuscript M114.588798 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 7, 2017 hp://w w w .jb.org/ D ow nladed from Disulfide-mutant SOD1 is active and has metal binding like wild type 2 attraction for superoxide, possibly explaining the lower rates. S. cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30-50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.