Abstract
S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.
Highlights
Erties of the cysteine [4]
We have demonstrated with atomic resolution the effects of S-nitrosylation on the x-ray crystal structure of blackfin tuna myoglobin
Structure of Blackfin Tuna Myoglobin—The structure of myoglobin isolated from blackfin tuna was determined to 0.91 Å resolution by molecular replacement
Summary
We have demonstrated with atomic resolution the effects of S-nitrosylation on the x-ray crystal structure of blackfin tuna myoglobin. We have identified conformational changes in this protein that are the direct result of the formation of the S-nitrosothiol group. Upon reduction with dithionite, the NO group is transferred from the surface-exposed Cys-10 to the reduced heme iron, showing the reversibility of the S–NO modification and the conformational changes. We have provided the first atomic resolution structure of nitric oxide bound to the heme iron of a globin protein, which allows the calculation of accurate geometric parameters for this important coordination complex
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