We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single- amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine- 87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low- spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild- type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87 suggested by the observation of a H/D sensitive resonance Raman line at 502 cm-1 that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to tire heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.
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