Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands

The role of heme ligand switching in redox regulation

Sangita Singh, Peter Madzelan, Jay Stasser, Colin L. Weeks, Donald F Becker, Thomas G. Spiro, James Penner-Hahn, Ruma Banerjee

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ∼20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52, respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424 nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449 nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is -350 ± 4 mV and is substantially lower than the value of -287 ± 2 mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.

Original languageEnglish (US)
Pages (from-to)689-697
Number of pages9
JournalJournal of Inorganic Biochemistry
Volume103
Issue number5
DOIs
StatePublished - May 1 2009

Fingerprint

Cystathionine
Heme
Oxidation-Reduction
Electronic structure
Modulation
Ligands
Pyridoxal Phosphate
Mutation
Catalytic Domain
Hemeproteins
Condensation reactions
Photolysis
Carbon Monoxide
Coulomb interactions
Static Electricity
Histidine
Electronic properties
Nitrogen
Switches
Oxygen

Keywords

  • Cystathionine beta-synthase
  • EXAFS
  • Heme
  • Resonance Raman spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands : The role of heme ligand switching in redox regulation. / Singh, Sangita; Madzelan, Peter; Stasser, Jay; Weeks, Colin L.; Becker, Donald F; Spiro, Thomas G.; Penner-Hahn, James; Banerjee, Ruma.

In: Journal of Inorganic Biochemistry, Vol. 103, No. 5, 01.05.2009, p. 689-697.

Research output: Contribution to journalArticle

Singh, Sangita ; Madzelan, Peter ; Stasser, Jay ; Weeks, Colin L. ; Becker, Donald F ; Spiro, Thomas G. ; Penner-Hahn, James ; Banerjee, Ruma. / Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands : The role of heme ligand switching in redox regulation. In: Journal of Inorganic Biochemistry. 2009 ; Vol. 103, No. 5. pp. 689-697.
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AU - Stasser, Jay

AU - Weeks, Colin L.

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AU - Spiro, Thomas G.

AU - Penner-Hahn, James

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AB - In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ∼20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52, respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424 nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449 nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is -350 ± 4 mV and is substantially lower than the value of -287 ± 2 mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.

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