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.
Results: The results revealed that the heme was stably associated to the protein under all denaturing
conditions with His117 playing the anchoring role. The studies also highlighted the possibility
of existence of a “molten globule” like intermediate at acidic pH in this exceptionally thermostable
globin. His117 and other key residues in the heme pocket play an indispensable role in imparting
significant polypeptide stability.
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.