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Olga Schröder,
Boris Bleijlevens, Thyra E de Jongh,
Zhujun Chen,
Tianshu Li,
Jörg Fischer,
Jochen Förster,
Cornelius G Friedrich,
Kimberly A Bagley,
Simon P J Albracht,
Wolfgang Lubitz
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ABSTRACT: Electron paramagnetic resonance (EPR) and Fourier transform IR studies on the soluble hydrogenase from Acidithiobacillus ferrooxidans are presented. In addition, detailed sequence analyses of the two subunits of the enzyme have been performed. They show that the enzyme belongs to a group of uptake [NiFe] hydrogenases typical for Cyanobacteria. The sequences have also a close relationship to those of the H(2)-sensor proteins, but clearly differ from those of standard [NiFe] hydrogenases. It is concluded that the structure of the catalytic centre is similar, but not identical, to that of known [NiFe] hydrogenases. The active site in the majority of oxidized enzyme molecules, 97% in cells and more than 50% in the purified enzyme, is EPR-silent. Upon contact with H(2) these sites remain EPR-silent and show only a limited IR response. Oxidized enzyme molecules with an EPR-detectable active site show a Ni(r)*-like EPR signal which is light-sensitive at cryogenic temperatures. This is a novelty in the field of [NiFe] hydrogenases. Reaction with H(2) converts these active sites to the well-known Ni(a)-C* state. Illumination below 160 K transforms this state into the Ni(a)-L* state. The reversal, in the dark at 200 K, proceeds via an intermediate Ni EPR signal only observed with the H(2)-sensor protein from Ralstonia eutropha. The EPR-silent active sites in as-isolated and H(2)-treated enzyme are also light-sensitive as observed by IR spectra at cryogenic temperatures. The possible origin of the light sensitivity is discussed. This study represents the first spectral characterization of an enzyme of the group of cyanobacterial uptake hydrogenases.
JBIC Journal of Biological Inorganic Chemistry 03/2007; 12(2):212-33. · 3.29 Impact Factor
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ABSTRACT: To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5-6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK( a,1)), an active form (pK( a,1)<pH<pK( a,2)), and an inactive high pH form (pH>pK( a,2)). The pK( a,1) values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme-phosphate complex (which should mimic the enzyme-substrate complex), the enzyme-fluoride complex, and the enzyme-fluoride-phosphate complex (which should mimic the ternary enzyme-substrate-hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.
JBIC Journal of Biological Inorganic Chemistry 08/2005; 10(5):550-63. · 3.29 Impact Factor
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Olga Schröder,
Boris Bleijlevens, Thyra E. de Jongh,
Zhujun J. Chen,
Tianshu S. Li,
Jörg Fischer,
Jochen Förster,
Cornelius G Friedrich,
Kimberly A Bagley,
Simon P J Albracht,
Wolfgang Lubitz
Journal of Biological Inorganic Chemistry, v.12, 212-233 (2007).
