Stability and Folding of the Unusually Stable Hemoglobin from Synechocystis is Subtly Optimized and Dependent on the Key Heme Pocket Residues

Abstract

Aims: The aim of our study is to understand the biophysical traits that govern the stability and folding of Synechocystis hemoglobin, a unique cyanobacterial globin that displays unusual traits not observed in any of the other globins discovered so far. Background: For the past few decades, classical hemoglobins such as vertebrate hemoglobin and myoglobin have been extensively studied to unravel the stability and folding mechanisms of hemoglobins. However, the expanding wealth of hemoglobins identified in all life forms with novel properties, like heme coordination chemistry and globin fold, have added complexity and challenges to the understanding of hemoglobin stability, which has not been adequately addressed. Here, we explored the unique truncated and hexacoordinate hemoglobin from the freshwater cyanobacterium Synechocystis sp. PCC 6803 known as “Synechocystis hemoglobin (SynHb)”. The “three histidines” linkages to heme are novel to this cyanobacterial hemoglobin. Objective: Mutational studies were employed to decipher the residues within the heme pocket that dictate the stability and folding of SynHb. Methods: Site-directed mutants of SynHb were generated and analyzed using a repertoire of spectroscopic and calorimetric tools. Conclusion: Synechocystis hemoglobin presents an important model system for investigations of protein folding and stability in general. The heme pocket residues influenced the folding and stability of SynHb in a very subtle and specific manner and may have been optimized to make this Hb the most stable known as of date. Other: The knowledge gained hereby about the influence of heme pocket amino acid side chains on stability and expression is currently being utilized to improve the stability of recombinant human Hbs for efficient use as oxygen delivery vehicles.