D Barber

Studies of cyanide binding to myeloperoxidase by electron paramagnetic resonance and magnetic circular dichroism spectroscopies

Abstract The techniques of magnetic circular dichroism (MCD) and absorbance spectroscopy have been used to monitor titrations of oxidized myeloperoxidase with CN − , at room temperature, over the Soret, visible and near-infrared regions of the spectrum. Electron paramagnetic resonance measurements have also been made between 10–30 K. The near-infrared MCD of the unbound enzyme possesses a bi-signate band at 1000 nm, similar to those seen with other high-spin haemoproteins. On addition of CN − this band is replaced by a positive band at 1500 nm, which is assigned to low-spin haem. This switch in spin state was also observed by EPR spectroscopy with high-spin signals at g 7.09 and 5.17 titrating away to low-spin signals at g 2.58, 2.33 and 1.81. Significant changes were apparent even at ratios of CN − /haem of less than one. Integrations of the EPR spectra bave been made to check absorbance spectrum extinction coefficients, in the absorbance and MCD spectra measured over the Soret and visible bands evidence was found for more than one CN − binding process. The MCD spectrum of cyanide-bound oxidized myeloperoxidase from 350 to 700 nm strongly suggests that the haem is closely related to a ring of the chlorin type.

The characterization and magnetic properties of the azide and imidazole derivatives of Pseudomonas nitrite reductase.

Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d1 with an affinity constant, Kaff = 360 M-1, whereas imidazole binds to both hemes c and d1 with kaff = 35 and 55 M-1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d1 are low-spin ferrihemes in both derivatives, although the epr of the heme d1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivatives in which heme d1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.

A characterization of ferric sulphaemoglobin

Abstract The kinetics of oxidation of ferro-sulphaemoglobin to ferri-sulphaemoglobin (met-sulphaemoglobin) by ferricyanide have been studied and the ligand binding properties of the product characterized. In contrast to normal haemogiobin, sulphaemoglobin reacts with ferricyanide at a rate of 4.5·103 M−1· s−1 and 1.3·103 M−1·s−1, at pH 6 and 8, in a simple fashion, suggesting equivalence of the α and β chains. The product, met-sulphaemoglobin, shows a high spin-low spin transition with a pK of 7.7 which is associated with the ionization of a single proton. The rate of spin-state change is so fast as to be rate-limited in the oxidation studies. Titration studies show that met-sulphaemoglobin binds the normal ferric haem ligands azide and cyanide both in an essentially non-cooperative manner. The equilibrium constants for azide and cyanide are 9.10-4 M and 3.5.10−4 M, respectively. Kinetic studies show that both ligands combine with the protein in a biphasic manner, the rates of combination varying considerably for both ligands, being 2. l02 M−1·s−1 and 3.3 M−1·s−1 for cyanide and 46 M−1·s−1 and 1.5 M−1·s-−1 for azide. A consideration of the measured dissociation rates of these ligands, taken with the combination rates, suggests that the simple ligand binding processes observed statically arise from a coincidence of the two associated kinetically observed processes. Magnetic circular dichroism spectra appear to substantiate the previous proposal for the structure of the haem of sulphaemoglobin as pseudo-chorin.

An investigation of the co-operative binding of carbon monoxide to the haemoglobin of the carpet shark Cephaloscyllium

1. Carpet Shark haemoglobin shows co-operative equilibrium CO binding at pH 7.0 (n = 1.8). 2. Flash photolysis shows that the T form of the protein is stabilised at low pH and in the presence of Inositol Hexaphosphate (I.H.P.). 3. At high pH the R form of the protein is stabilised and exhibits kinetic heterogeneity, assigned to different chain reactivities. 4. Stopped flow and flash photolysis under various conditions allows estimates to be made of the association and dissociation rates of CO binding for the two forms of the protein. 5. The functionality of the protein, with regard to CO binding, can be described by a modified two state model.

An investigation of the allosteric functioning of the haemoglobin of the cane toad, Bufo marinus

1. Cane toad haemoglobin shows co-operative equilibrium CO binding at pH 7.0 with n = 2.6. 2. Flash photolysis studies show that at high pH the R form of the protein is stabilised whilst low pH and the presence of IHP favours the T form. 3. The combination of stopped-flow and flash photolysis measurements allows estimation of the association and dissociation rates of CO binding for the two forms of the protein. 4. The protein functioning can be described by a modified two state model and mathematical modelling allows the estimation of the allosteric equilibrium constant for this haemoglobin.