Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: Evidence for ligation of tyrosine-63 (B10) to the heme

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-ami no acid mutants by EPR, optical absorbance, and resonance Raman spectroscop y. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are su ggested 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 th e Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protei n ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spi n 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 observ ation of a H/D sensitive resonance Raman line at 502 cm(-1) that is tentati vely assigned as a vibrational mode of the Fe-O bond between the iron and t he tyrosinate. We propose that the transition from the high-spin to the low -spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.