Reversible S-nitrosylation in an engineered azurin.
S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper-thiolate bond in an engineered copper centre in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)-S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Shiliang
S
Tian
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Jing
J
Liu
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Ryan E
RE
Cowley
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Parisa
P
Hosseinzadeh
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Nicholas M
NM
Marshall
Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Yang
0000-0002-0784-9715
Y
Yu
Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.
Howard
H
Robinson
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Mark J
MJ
Nilges
Institute of Environmental Health, Oregon Health &Sciences University, Portland, Oregon 97239, USA.
Ninian J
NJ
Blackburn
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Edward I
EI
Solomon
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.|Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.|Center of Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Yi
Y
Lu
D001400:Azurin / Q000138:chemical synthesis* / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D003545:Cysteine; D006860:Hydrogen Bonding; D008667:Metalloproteins / Q000737:chemistry; D009569:Nitric Oxide / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000378:metabolism
D008667:Metalloproteins; D026403:S-Nitrosothiols; D001400:Azurin; D009569:Nitric Oxide; D003300:Copper; D003545:Cysteine