title: Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme.
abstract: 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 the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Das
authors.forename: M
authors.initials: M
authors.lastname: Couture
authors.forename: H C
authors.initials: HC
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Peisach
authors.forename: D L
authors.initials: DL
authors.lastname: Rousseau
authors.forename: B A
authors.initials: BA
authors.lastname: Wittenberg
authors.forename: J B
authors.initials: JB
authors.lastname: Wittenberg
authors.forename: M
authors.initials: M
authors.lastname: Guertin
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002696:Chlamydomonas / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
chemical_list: D005290:Ferric Compounds; D006454:Hemoglobins; D008024:Ligands; D014443:Tyrosine; D006418:Heme