Primary and Secondary Coordination Sphere Effects on the Structure and Function of S-Nitrosylating Azurin

Abstract

Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact of SCS on reactivity is much less understood. A primary example is how copper proteins can promote S-nitrosylation (SNO), which is one of the most important dynamic post-translational modifications, and is crucial in regulating nitric oxide storage and transportation. Specifically, the factors that instill CuII with S-nitrosylating capabilities and modulate activity are not well understood. To address this issue, we investigated the influence of the primary and secondary coordination sphere on CuII-catalyzed S-nitrosylation by developing a series of azurin variants with varying catalytic capabilities. We have employed a multidimensional approach involving electronic absorption, S and Cu K-edge XAS, EPR, and resonance Raman spectroscopies together with QM/MM computational analysis to examine the relationships between structure and molecular mechanism in this reaction. Our findings have revealed that kinetic competency is correlated with three balancing factors, namely Cu–S bond strength, Cu spin localization, and relative S­(ps) vs S­(pp) contributions to the ground state. Together, these results support a reaction pathway that proceeds through the attack of the Cu–S bond rather than electrophilic addition to CuII or radical attack of SCys. The insights gained from this work provide not only a deeper understanding of SNO in biology but also a basis for designing artificial and tunable SNO enzymes to regulate NO and prevent diseases due to SNO dysregulation.

HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens

“…Specifically, models of WTAz and F114PAz were based directly on their respective crystal structures, while models of N47SAz and F114NAz were based on the structure of the F114N/N47SAz double mutant, as edited in Pymol 2.4.0. All QM/MM calculations were performed according to previous protocol, using ASH, which provides an interface for the QM program ORCA, , version 5.0.3, with the MM program OpenMM . MM calculations employed the CHARMM36 force field.…”