Cu/Zn coordination in protein structure-function relationships

Abstract

The evolution and adaptation of proteins across diverse environments present a fundamental challenge in understanding how homologous proteins preserve catalytic activity while optimizing for specific physiological conditions. To address this, we compare Cu/Zn superoxide dismutase from human (hSOD1) and Mycobacterium tuberculosis (MtSOD). Cu/Zn SOD are abundant across all domains of life and display catalytic activity near the diffusion limit, making them ideal models for studying sequence adaptation under environmental pressure. Sequence analysis reveals significant divergence, particularly in metal-binding regions, with MtSOD lacking residues essential for Zn2+ coordination. Structural characterization demonstrate that MtSOD compensates for this loss through rigid loop rearrangements and altered dimer interface, preserving stability and catalytic activity. Pyrogallol assays demonstrated that while hSOD1 strictly requires both Cu²⁺ and Zn²⁺ for activity, MtSOD retains full catalytic efficiency with Cu²⁺ alone. Despite sharing less than 20% sequence identity, they exhibit a highly conserved monomeric structural homology, underscoring the conservation of the catalytic fold. Our results show that while hSOD1 and MtSOD share a conserved fold and catalytic activity, they employ distinct molecular strategies to suit their environments. hSOD1 exhibits tighter core packing, higher stability (ΔG = 5.70 ± 0.21 kcal/mol), and an optimized surface charge distribution for the stable cytosolic environment of human cells. In contrast, MtSOD retains catalytic efficiency but has a more dynamic core, reduced stability (ΔG = 2.47 ± 0.10 kcal/mol), and diminished Zn²⁺ dependence, reflecting adaptation to variable and hostile conditions. These differences highlight how sequence evolution fine-tunes stability, packing, and charge while preserving overall structure and function under distinct environmental pressures.