Nature spent millions of years to evolve electron transfer proteins that span a wide range of reduction potentials (E°′) under physiological conditions, from −1 V to 1 V vs. SHE [1]. Understanding the rules that govern such tuning within similar classes of metalloproteins enables scientists to rationally tune the E°′ of their catalysts without changing the active site. An ultimate test of such understanding is to achieve the entire range of E°′ within a single protein, a feat that has not been achieved yet. We have recently achieved the goal of designing azurin to cover a range from +970 mV to −954 mV vs. SHE by mutating only five residues and using two metal ions (copper and nickel) [2]. Armed with this knowledge, we have used our ability of rational designing the primary coordination sphere and tuning its E°′ by deleting a hydrogen bond in the secondary coordination sphere to engineer the copper center in azurin to achieve reversible insertion of NO into a copper–thiolate bond [3]. S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalyzed by heme proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Therefore, 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. Such a fast, efficient and reversible S-nitrosylation under physiologically relevant conditions discovered in the engineered azurin reported here suggests that it is possible that nature may use such a reaction by copper proteins for biological functions such as NO delivery or regulation.

Design of azurin that spans the entire 2V range of physiological redox potentials and its application in engineering a copper protein that performs fast and reversible S-nitrosylation

253rd National Meeting (2017)

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