Ninian J. Blackburn

Dopamine-β-hydroxylase: Evidence for binuclear copper sites

Abstract Copper has been progressively removed from dopamine-β-hydroxylase by incubation with chelex resin. The relationship between the activity (A) and the copper/protein ratio (r) has been shown to be of the form A ⊥ r 2 which implies that the enzyme catalysed reaction is second order with respect to protein bound copper. These results support a model for the catalytic mechanism involving binuclear copper sites rather than the four copper atoms in the enzyme acting independently.

Studies on a model copper mono-oxygenase system: Peroxo-CuII binuclear intermediates in the hydroxylation of an aromatic ring

Hydrogen peroxide reacts with a CuII complex of a m-xylyl binucleating ligand with concomitant hydroxylation to give a phenoxo-bridged complex (previously found as the product of the reaction of dioxygen with the CuI analogue) whereas a newly synthesized mononuclear analogue is unreactive, thereby implicating µ-peroxo–CuII intermediates in the hydroxylation reaction.

EXAFS Studies of Zinc and Copper in Chemical and Biochemical Systems

Recent X-ray absorption spectroscopic studies we have undertaken at the Daresbury Laboratory using the dedicated Synchrotron Radiation Source have included investigations of the copper and zinc sites of mammalian liver metallothioneins and bovine erythrocyte superoxide dismutase. Corresponding studies have been accomplished for several copper and zinc compounds, the structure of each having been established by X-ray crystallography and shown to have some relevance to that of the metals in one or other of the above proteins.

EXAFS Studies of Non-Blue Copper Proteins: Superoxide Dimutase, Dopamine-Monooxygenase and Monoamine Oxidase

The copper containing enzymes superoxide dismutase, dopamine monooxygenase and plasma amine oxidase while functionally diverse are often classified together as ‘non-blue’ or ‘type 2’ on the basis of the similarity of their optical and EPR spectroscopic properties to those of simple mononuclear complexes[1]. X-ray absorption spectroscopy offers a further method of examining the structural relationships within this class of copper proteins. In this paper we summarise the results of EXAFS studies, which provide evidence of structural homologies and have identified imidazole coordination in all three enzyme systems. In the case of native superoxide dismutase where a crystal structure is available to 2A resolution[2, 3], comparison of EXAFS and crystal lographic coordinates using molecular graphics has provided extra insights into the local structure of the active site.

EXAFS studies of copper and zinc in metallothionein and bovine superoxide dismutase

We have accomplished the measurement and interpretation of the copper and zinc K-edge EXAFS for these elements in two protein environments; metallothionein, from a variety of sources, and bovine erythrocyte superoxide dismutase. Metallothioneins are a ubiquitous class of proteins which are believed to play important roles in the control of zinc and copper metabolism and in the detoxification of metals, such as cadmium and mercury. Each of these proteins is characterized by its low molecular weight (∼6,000), a high cysteinyl content, and the ability to bind substantial amounts of metal ions, such as Cd, Zn, Cu, Ag, Co and Hg. Results obtained from a variety of spectroscopic studies clearly indicate that the metal ions are bound to the protein via the sulphur atoms of the cysteinyl residues. However, a definitive structural characterization of a metallothionein has not yet been achieved. To provide further information concerning the details of metal attachment to metallothionein, we have recorded the copper and zinc K-edges for pig liver metallothionein containing copper and zinc, and the K-edge of zinc for sheep liver metallothionein 2 containing zinc, and rabbit liver metallothionein containing zinc or zinc and cadmium. Freeze-dried samples were employed and X-ray absorption spectra were recorded for these materials at room temperature and 77K. For zinc, the EXAFS data are consistent with back-scattering from, essentially, a single shell of our sulphur atoms at ca. 2.28 A. The chemical interpretation of this information is that zinc is bound by the sulphur atom of four cysteinyl groups, as Zn(Scys)4. Similar conclusions have been reached for the immediate environment of the copper atoms in pig liver metallothionein containing ca. six copper and two zinc atoms per protein molecule. The CuS distance is ca. 2.22 A and there appear to be three sulphur atoms at this distance. An assessment of the quality of interpretation of the EXAFS data obtained for zinc and copper in metallothionein has been obtained by graphically displaying the variation of the function rg = 1gSk[k2(ψT(k) − ψE(k))]2 for different values of the parameters used to interpret the EXAFS data. The reciprocal of the simple least-squares function was chosen since it produces clearer visual displays than the function itself; thus, the optimum interpretation of the experimental data is when rg is maximised. An iterative procedure was adopted, refining (R and E0) and (N and α) sequentially, until reasonable self-consistency was obtained. The final value of R (ZnS) was obtained by plotting a slice through the rgversus (R, E0) profile at the optimum value of E0; the uncertainty in this value being taken as the width of this peak at 70% height. Bovine erythrocyte dismutase has been the subject of a large number of investigations and much of this work has been stimulated by the determination of the crystal structure to 3 A resolution. However, this structure alone does not contain the detailed information needed to explain aspects of the enzymes mechanism as the resolution is too low to detect the subtle structural features which presumably control the activity. Therefore, we have undertaken an X-ray absorption spectroscopic study of the metal centres in this superoxide dismutase, in its native, reduced, and azide bound forms. Data was recorded for freeze dried material and aqueous solutions of the protein. The similarities in the profile of the copper K-edge EXAFS of native superoxide dismutase and those obtained for [Zn(imidazole)4] [ClO4]2 and [Cu(imidazole)4(NO3)2], indicate that, in this form of the enzyme, the copper is co-ordinated by four essentially equivalent imidazole groups, with CuNα distances of ca. 2.00 A. The CU⋯Cβ and Cu⋯Cγ(Nγ) distances follow from the CuNα value. A similar environment is found about copper in the azide bound form but reduction causes a significant structural change at this atom. The EXAFS data obtained for the zinc site of native superoxide dismutase are reasonably reproduced by an environment comprising one oxygen atom, presumably from the asparate carboxylate group, at ca. 1.92 A and one and two imidazole groups with ZnN distances of ca. 2.06 and 1.97 A, respectively. The geometry about this atom is not changed in any significant respect in the azide bound and reduced forms of the enzyme. These results will be discussed with reference to EXAFS data for a variety of copper and zinc complexes of a known structure. Attempts to determine the geometry of the sulphur atoms about the metal atoms of metallothionein from XANES data will also be described.

Fluorescence EXAFS of biological systems at SRS

Abstract A fluorescence detection system consisting of five scintillation detectors has been commissioned recently on an EXAFS station at the worlds first dedicated X-ray synchrotron radiation source (SRS). This facility has been established to measure the X-ray fluorescence excitation spectrum of specimens containing small amounts of metal atoms with absorption edges between 1 A and 3.1 A. Results recently obtained on copper containing enzymes are presented.