H.-F. Nolting

Evidence for discrete Cd(SCys)4 units in cadmium phytochelatin complexes from EXAFS spectroscopy

The principal structural features of multinuclear complexes of cadmium(II) with the plant-peptide phytochelatin have been deduced from Cd-EXAFS (EXAFS = extended X-ray absorption fine structure) data: (i) Cd(SCys)4 coordination, (ii) in contrast to Cd-metallothioneins, no formation of cadmium ‘clusters’ and (iii) non-coordinating carboxylate groups.

X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the Zn K edge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess beta-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboom et al. (1997), Curr. Biol. 7, 739-746; (2000), J. Biomol. NMR, 18, 119-28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations with EXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 A (Debye-Waller-type factor 0.009 A(2)) and two imidazole ligands with the N atoms at 1.99 A (Debye-Waller-type factor 0.005 A(2)). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the Volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn-S and Zn-N distances of 2.3 A and 2.0 A, respectively).

The first diferric model compound for the uteroferrin–arsenato complex

The X-ray crystal structure and EXAFS measurements are reported for a newly synthesised unsymmetrical dinuclear iron(III) complex representing the first diferric compound with a single bridging arsenato group.

Energy resolution using delay line detectors

Abstract The use of multiwire proportional chambers (MWPC) with delay line readout for applications involving energy resolution is considered. The main features of this approach and some possible applications are presented. Its extension to neutron detection using MWPC or scintillators with position sensitive photomultipliers is also illustrated.

Cobalt X-ray absorption spectroscopy of glucose isomerase

Introduction Glucose isomerases catalyze the isomerization of D-glucose to D-fructose and of D-xylose to D-xylulose. They are of considerable interest in the industrial production of high-fructose corn syrup. Glucose isomerase from Streptomyces albus (GI) is a tetramer (Mr = 172.000) with four identical subunits [1,2]. Each subunit contains two distinct metal bindin of Co2+, Mn f sites, a tight binding site (B-site) and a weaker binding site (A-site). Addition + or Mg2’ * increases the catalytic activity as well as the stability of the enzyme.

Specimen preparation of air sensitive materials for X-ray absorption measurements

Abstract An improved technique for the preparation of solid specimens suitable for X-ray absorption measurements is described which is especially useful for air or water sensitive materials. The finely powdered materials are enclosed in a polymer matrix. Homogeneous, stable, and uniformly thick films can be obtained by this method. The capabilities of the technique have been tested within a series of iron and cobalt compounds.

EXAFS studies on Iron(III) sorbitol complexes

Abstract Complexes of Fe(III) with sorbitol in alkaline aqueous solutions have been investigated by EXAFS spectroscopy. The results strongly support the formation of a mononuclear complex at pH 14, a dinuclear complex at pH 13 and oligonuclear species at pH 10–13. The EXAFS spectra were recorded at 70 K in the absorption mode.

XAFS of human tyrosine hydroxylase

Abstract Tyrosine hydroxylase (TH) catalyses the rate-limiting step (hydroxylation of tyrosine to form dihydroxyphenylalanine) in the biosynthetic pathway leading to the catecholamines dopamine, noradrenaline and adrenaline. The human enzyme (hTH) is present in four isoforms, generated by splicing of pre-mRNA. The purified apoenzyme (metal free) binds stoichiometric amounts of iron. The incorporation of Fe(II) results in a rapid and up to 40-fold increase of activity [1]. Besides the coordination of the metal centers in native enzyme we studied the purported inhibition of TH by its immediate products. So we analysed Fe-hTH isoform 1 native as well as oxidized with dopamine and Co-hTH isoform 2.

X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)

Abstract Purple acid phosphatase from red kidney beans (KBP) catalyzes the hydrolysis of activated phosphoric acid monoesters and contains a heterodinuclear Fe(III)Zn(II) core in its active site. Iron K-edge X-ray absorption data have been obtained for the native enzyme and for a metal exchanged derivative, where Zn(II) was substituted by Fe(III). The environment of the native enzyme consists of 2.5 O/N at 1.91 A, 3 O/N at 2.09 A, and 1 Zn at 4.05 A. For the metal exchanged form we obtained 2.5 O/N at 1.94 A, 2.5 O/N at 2.09 A, and 1 Fe at 3.79 A.

A Structural Model for the Soybean Lipoxygenase-1 Iron Site

In this communication the striking similarity between the EXAFS of soybean lipoxygenase-1 and that of a methanol-solution of a well-characterized iron-polyimidazole model compound is reported. This similarity is a strong corroboration of the structure proposed earlier for the non-heme iron active site in lipoxygenase (S. Navaratnam, M.C. Feiters, M. Al-Hakim, J.C. Allen, G.A. Veldink, J.F.G. Vliegenthart, Biochim. Biophys. Acta, 1988, 956, 70)