Protein Dynamics Govern the Oxyferrous State Lifetime of an Artificial Oxygen Transport Protein
It has long been known that the alteration of protein side chains which occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here we demonstrate that the rate of dynamically-driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically-driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity which limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins. SignificanceThis communication sheds light on one of the more controversial areas in protein folding and design: the dynamic nature of the hydrophobic core and its relationship to metalloprotein function, in particular the relationship between dynamic solvent penetration into the protein core and the stability of metalloenzyme intermediates. We demonstrate that the basic tetrameric scaffold that is the classic helical bundle model for cofactor binding and activation can be easily upgraded to a more rigid, less dynamic, single chain helical bundle by merely taking the same helical sequences and converting it to a single chain protein connected by simple, nonoptimized glycine-rich loops. Importantly, our results explain the decades-long history of failure in the design of proteins capable of stably forming an oxyferrous state - the requirement for a protein large enough to protect the heme porphyrin surface with both structural specificity and sufficient structural rigidity to restrict water penetration into the protein core. Finally, we believe this is the first use of Deep UV Resonance Raman spectroscopy to monitor dynamic water penetration in a functional protein. This method may prove useful moving forward to many research groups.