Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Parameciu m caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacteri um tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the pu rification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contain s Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxy gen dissociation rate measured, 0.011 s(-1), is among the smallest known an d is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and r esonance Raman spectroscopic study of the structure of the heme pocket of S ynechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5-10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid se quence comparison, is involved in the formation of the ferric and ferrous 6 -coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form con tains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild typ e Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.