Structure of the Native Cysteine-Sulfenic Acid Redox Center of Enterococcal NADH Peroxidase Refined at 2.8 Å Resolution

In order to obtain the crystal structure of the flavoprotein NADH peroxidase with its native Cys42-sulfenic acid redox center, a strategy combining reduced exposure of crystals to ambient oxygen and data collection at −160 °C was applied. The structure of the native enzyme to 2.8 Å resolution is described; these results conclusively establish the existence of the Cys42-sulfenic acid as the functional non-flavin redox center of the peroxidase and provide the first structure for any naturally occurring protein-sulfenic acid. The Cys42-sulfenic acid atoms Cα−Cβ−Sγ−O roughly define a planar arrangement which is stacked parallel to the si face of the FAD isoalloxazine and positions the sulfenyl oxygen atom only 3.3 Å from FAD-C4A. His10-Nε2 contributes a hydrogen bond to the sulfenic acid oxygen, at a distance of 3.2 Å. Although one oxygen atom (OX1) of the non-native Cys42-sulfonic acid derivative identified in the earlier wild-type peroxidase structure was taken to represent the native Cys42-sulfenic acid oxygen [Stehle, T., Ahmed, S. A., Claiborne, A., & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325−1344], this structure shows that the sulfenic acid oxygen does not occupy this position, nor is it hydrogen-bonded to Cys42-N as was OX1. Comparison of the native Cys42-sulfenic acid structure with that of two-electron reduced glutathione reductase provides an insight into the sulfenic acid FAD charge-transfer interaction observed with both wild-type and His10 mutant peroxidases. A model of the E·NADH intermediate recently observed in stopped-flow analyses of the enzyme [Crane, E. J., III, Parsonage, D., Poole, L. B., & Claiborne, A. (1995) Biochemistry 34, 14114−14124] has also been generated to assist in analyzing the chemical mechanism of sulfenic acid reduction.