Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket
2000
Couture, Manon | Das, Tapan Kanti | Savard, PierreâYves | Ouellet, Yannick | Wittenberg, Jonathan B. | Wittenberg, Beatrice A | Rousseau, Denis L. | Guertin, Michel
A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Feâprotoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011âs−1, is among the smallest known and is four orders of magnitude smaller than the rate measured for N.âcommune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6âcoordinate and lowâspin 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 aminoâacid sequence comparison, is involved in the formation of the ferric and ferrous 6âcoordinate lowâspin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5âcoordinate and highâspin and the ferric form contains a 6âcoordinate highâspin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.
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