Magneto-optical spectroscopic studies of multi-heme enzymes
type: CreativeWork
description: Cytochrome cd1 (cd1) is a soluble, diheme enzyme located in the periplasm of denitrifying bacteria that catalyses the one-electron reduction of nitrite ion to nitric oxide. Paracoccus pantotrophus cd1 undergoes an unusual coordinated ligand switch upon reduction to the diferrous state, whereby a distal tyrosine ligand dissociates from the active site d1-heme, while the ligands at the c-heme switch from bis-histidinyl to histidine/methionine. The processes that take place following re-oxidation of this enzyme have been studied using a combination of electronic absorption, electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. Rebinding of tyrosine to the d1-heme, and loss of the bound product occurs concomitantly with a switch back to bis-histidinyl ligation at the c-heme at a rate of ~ 0.24 min−1 at pH 6.50. However, this does not represent a return to the “as-prepared” conformation: the two c-heme histidine ligands are in a novel ‘parallel’ conformation. MCD spectroscopy has been used to characterise the state formed when reduced cd1 reacts with stoichiometric nitrite ion, a one-electron oxidant. A pH-dependent equilibrium between two forms of the semi-reduced product-bound enzyme is observed, [d12+-NO c3+] ⇌ [d12+-NO+ c2+]. A pKa of 7.10 is assigned to protonation of a distal histidine residue. It is proposed that in the protonated form His-H+, this residue stabilises the d12+-NO form of the active site. Ligand binding to the enzyme SoxAX, a tri-heme thiosulfate oxidase from Rhodovulum sulfidophilum has been studied using a combination of electronic absorbance and EPR spectroscopies. The enzyme proved to be resistant to ligand binding in the ‘as prepared’ state. Only by redox cycling in presence of excess concentrations of potassium cyanide (KCN) has it been possible to achieve ligand binding to at least one of the three c-type hemes.
abstract: Cytochrome cd1 (cd1) is a soluble, diheme enzyme located in the periplasm of denitrifying bacteria that catalyses the one-electron reduction of nitrite ion to nitric oxide. Paracoccus pantotrophus cd1 undergoes an unusual coordinated ligand switch upon reduction to the diferrous state, whereby a distal tyrosine ligand dissociates from the active site d1-heme, while the ligands at the c-heme switch from bis-histidinyl to histidine/methionine. The processes that take place following re-oxidation of this enzyme have been studied using a combination of electronic absorption, electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. Rebinding of tyrosine to the d1-heme, and loss of the bound product occurs concomitantly with a switch back to bis-histidinyl ligation at the c-heme at a rate of ~ 0.24 min−1 at pH 6.50. However, this does not represent a return to the “as-prepared” conformation: the two c-heme histidine ligands are in a novel ‘parallel’ conformation. MCD spectroscopy has been used to characterise the state formed when reduced cd1 reacts with stoichiometric nitrite ion, a one-electron oxidant. A pH-dependent equilibrium between two forms of the semi-reduced product-bound enzyme is observed, [d12+-NO c3+] ⇌ [d12+-NO+ c2+]. A pKa of 7.10 is assigned to protonation of a distal histidine residue. It is proposed that in the protonated form His-H+, this residue stabilises the d12+-NO form of the active site. Ligand binding to the enzyme SoxAX, a tri-heme thiosulfate oxidase from Rhodovulum sulfidophilum has been studied using a combination of electronic absorbance and EPR spectroscopies. The enzyme proved to be resistant to ligand binding in the ‘as prepared’ state. Only by redox cycling in presence of excess concentrations of potassium cyanide (KCN) has it been possible to achieve ligand binding to at least one of the three c-type hemes.
author: Haynes, Kate
language: en
date published: 2013-06
date modified: 2026-07-24T02:11:54Z
provider: University of East Anglia
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of East Anglia