Reversible S-nitrosylation in an engineered azurin

Abstract

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.

Figure 1. Geometries of Cu(II) binding sites and Fourier transform of EXAFS data with fitting

a, WTAz (PDB: 4AZU), b, nitrosocyanin (PDB: 1IBY) and c, M121H/H46EAz (PDB: 4WKX). The Cu(II)-binding site in Cu(II)-M121H/H46EAz adopted a more distorted tetrahedral geometry compared to WTAz. The Cu-S(Cys112) distance in Cu(II)-M121H/H46EAz was 2.28 Å, longer than the ∼ 2.1 Å in blue copper proteins, but comparable to that in nitrosocyanin (2.29 Å). An exogenous water was at 3.78 Å from the Cu(II) within hydrogen bonding distance of the coordinating O of Glu46 (2.56 Å) in Cu(II)-M121H/H46EAz. Fourier transform and EXAFS (inset) for d, Cu(II)-M121H/H46E/F114PAz, e, Cu(I)-M121H/H46E/F114PAz and f, NO treated Cu(II)-M121H/H46E/F114PAz. The NO treated Cu(II)-M121H/H46E/F114PAz has different EXAFS pattern from Cu(I)-M121H/H46E/F114PAz, indicating the process was not simple reduction from Cu(II) to Cu(I). The NO treated Cu(II)-M121H/H46E/F114PAz was best fitted when the Cys ligand was moved to 3.98 Å and a new N ligand was added at 2.83 Å, suggesting that the weak interaction between Cu(I) and S-nitrosothiol is through the N-atom. Experimental data are shown as solid black lines, and the simulationsas red lines. Copper atoms are green; oxygen atoms are red; sulfur atoms are yellow; nitrogen atoms are blue; carbon atoms are cyan.

Figure 2. UV-Vis absorption, Mass spectrometric and EPR spectroscopic characterization of S-nitrosylation in engineered red copper protein

a, The UV-Vis spectra of Cu(II)-WTAz, Cu(II)-M121H/H46EAz and Cu(II)-M121H/H46E/F114PAz. The S(Cys)→Cu(II) LMCT band of Cu(II)-M121H/H46E/F114PAz was at 390 nm, blue shifted compared to WTAz, but identical to nitrosocyanin. b, Kinetic UV-Vis spectra of 110 μM Cu(II)-M121H/H46E/F114PAz reacting with 1 equivalent of DEA NONOate in 50 mM Mes buffer at pH 6. A spectrum was recorded every 10 s. Upon NO addition, immediate bleaching of the 390 nm peak and an isosbestic point at 347 nm were observed. c, ESI-MS before (black) and after (red) NONOate addition. A mass increase of +29 Da compared to apo-M121H/H46E/F114PAz supports S-nitrosylation of the protein. d, X-band EPR spectra of Cu(II)-M121H/H46E/F114PAz reacting with different amounts of NO. Inset: EPR integration as a function of NO equivalents. Decreasing signal intensity with increasing concentration of NO indicates the formation of an EPR silent species.

Figure 3. Electrophilic and radical pathways for (Cys)S-NO bond formation and possible isomers of the Cu(I)/(Cys)S-NO product

In pathway 1, coordination of NO to Cu(II) affords a singlet five-coordinate {CuNO} species. The intramolecular electrophilic attack of NO⁺ on S(Cys) occurs and forms η²-(Cys)S-NO product. In pathway 2, NO reacts directly with S(Cys) via radical coupling without the formation of a {CuNO} intermediate. All energies are reported as free energies (ΔG and ΔG^‡) at 298 K relative to the Cu(II) starting complex and free NO.

Figure 4. Effect of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo₃ oxidase

Time courses of O₂ reduction catalyzed by 10 nM cytochrome bo₃ oxidase, 100 μM ubiquinol-1 and 5 mM DTT (A) in the absence of NO, (B) in the presence of 5 μM NO, (C) 5 μM NO and 1 μM Cu(II)-M121H/H46E/F114PAz, (D) 5 μM NO and 2 μM Cu(II)-M121H/H46E/F114PAz, (E) 5 μM NO and 3 μM Cu(II)-M121H/H46E/F114PAz, (F) 5 μM NO and 4 μM Cu(II)-M121H/H46E/F114PAz, or (G) 5 μM NO and 5 μM Cu(II)-M121H/H46E/F114PAz. Inset: O₂ consumption rates derived from the slopes of initial linear regions in O₂ concentration curves. All the measurements were repeated three times and error bars indicated standard deviation. In the presence of NO, the O₂ reduction rate of cytochrome bo₃ oxidase decreased. The addition of Cu(II)-M121H/H46E/F114PAz to the above solution rescued the NO inhibition through NO sequestration, recovering 71% activity of cytochrome bo₃ oxidase.