Exogenous ligand binding kinetics of globin X.

Abstract

Intrinsically hexa-coordinate vertebrate globins have been shown to carry various cellular functions including neuroprotective and antitumor properties but the precise mechanism of their action remains unclear. Here we focus on the molecular mechanism of diatomic ligand interactions with globin X (GbX) which is a hexa-coordinate globin expressed in lower vertebrate such as fish, amphibians, and reptiles. GbX was proposed to have a variety of enzymatic activities including nitrite reductase, and nitric oxide dioxygenase along with the ability to bind several exogeneous ligands such as O2, CO, and CN-. The wide array of enzymatic functions present in GbX along with its low intracellular concentration in vivo and its hexa-coordination has led to the theory that GbX may serve to scavenge reactive oxygen and nitrogen species. Another interesting feature of GbX is the presence of an internal disulfide bond between residues Cys63 and Cys141 and myristoylation of its N-terminus with an as of yet unknown role in the functional properties of GbX. In this study interactions of exogenous ligands, CO and CN- with GbXWT and GbXH90V and GbXC60A mutants were investigated by combining in vitro and in silica approaches. The results of flash photolysis and stop flow experiments demonstrate multiphasic ligand rebinding to all three constructs with the slowest ligand binding to GbXWT. Time-resolved UV-Vis measurements also revealed multiphasic binding of CN- to GbX. The structures of GbX constructs were determined using homology modeling and subsequent MD simulations and the possible ligand migration pathways were probed using Implicit Ligand Sampling.