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

Tapan Kanti Das, Manon Couture, H. Caroline Lee, Jack Peisach, Denis L. Rousseau, Beatrice A. Wittenberg, Jonathan B. Wittenberg, Michel Guertin

Research output: Contribution to journalArticle

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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 tire heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.

Original languageEnglish (US)
Pages (from-to)15360-15368
Number of pages9
JournalBiochemistry
Volume38
Issue number46
DOIs
StatePublished - Nov 16 1999

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Chlamydomonas
Chloroplasts
Heme
Ligation
Tyrosine
Hemoglobins
Ligands
Lysine
Proteins
Paramagnetic resonance
Oxygen
Amino Acids
Deprotonation
Globins
Raman Spectrum Analysis
Cytochromes c
Glutamine
Tires
Raman spectroscopy
Protons

ASJC Scopus subject areas

  • Biochemistry

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Identification of the ligands to the ferric heme of chlamydomonas chloroplast hemoglobin : Evidence for ligation of tyrosine-63 (B10) to the heme. / Das, Tapan Kanti; Couture, Manon; Lee, H. Caroline; Peisach, Jack; Rousseau, Denis L.; Wittenberg, Beatrice A.; Wittenberg, Jonathan B.; Guertin, Michel.

In: Biochemistry, Vol. 38, No. 46, 16.11.1999, p. 15360-15368.

Research output: Contribution to journalArticle

Das, Tapan Kanti ; Couture, Manon ; Lee, H. Caroline ; Peisach, Jack ; Rousseau, Denis L. ; Wittenberg, Beatrice A. ; Wittenberg, Jonathan B. ; Guertin, Michel. / Identification of the ligands to the ferric heme of chlamydomonas chloroplast hemoglobin : Evidence for ligation of tyrosine-63 (B10) to the heme. In: Biochemistry. 1999 ; Vol. 38, No. 46. pp. 15360-15368.
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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 tire heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.",
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T1 - Identification of the ligands to the ferric heme of chlamydomonas chloroplast hemoglobin

T2 - Evidence for ligation of tyrosine-63 (B10) to the heme

AU - Das, Tapan Kanti

AU - Couture, Manon

AU - Lee, H. Caroline

AU - Peisach, Jack

AU - Rousseau, Denis L.

AU - Wittenberg, Beatrice A.

AU - Wittenberg, Jonathan B.

AU - Guertin, Michel

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N2 - 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.

AB - 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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