Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum

Infrared Studies on the Interaction of Carbon Monoxide with Divalent Nickel in Hydrogenase from Chromatium vinosum

“…This is demonstrated in Figure 1; incubation with ascorbate/PMS for 1 h at room temperature under an Ar atmosphere only affected a small part of the enzyme molecules, in which the Ni(II1) species were spin coupled to the 3Fe-cluster. The coupling disappeared as a result of reduction (Coremans et al, 1992a), and a very slow reduction of the 3Fe-cluster set in (not shown). When monitored at 50 K, Wavenumber (cm" ) FIGURE 2: Infrared absorbance spectrum of untreated, oxidized hydrogenase from C. vinosum at 20 K. EPR measurements indicated that this untreated enzyme contained 85% Nir(III) and 15% Ni.-(111).…”

“…The intensity of the Ni,-(111) signal was not significantly affected in either case. These observations can be explained by assuming that C O can bind specifically toNi,(II), thereby increasing its apparent midpoint potential [-115 mV at pH = 8.0 (Coremans et al, 1992a)J to such a value that excess ascorbate can reduce it and resulting in an EPR silent Ni(I1)-CO species. The present experiments suggest that the apparent midpoint potential of the Ni,(III)/ Ni,(II) couple under 1 bar of C O is well above 0 mV [in fact, other experiments (J. W. Van der Zwaan, M. Chen, and S. P. J. Albracht, unpublished observations) indicated a potential higher than 350 mV].…”

“…In both the inactive forms of hydrogenase from C. vinosum, Ni,(III) and Niu(III), a n = 1 transition, as evidenced by the disappearance of the S = '/z EPR signals associated with the Ni, could be observed in the presence of mediating dyes at pH = 8 and 30 OC at exactly the same midpoint potential (Eo' = -1 15 mV) (Coremans et al, 1992a). This transition was reversible and ascribed to a Ni(III)/Ni(II) transition for both the ready and unready forms of the enzyme.…”

Further Characterization of the Spin Coupling Observed in Oxidized Hydrogenase from Chromatium vinosum. A Moessbauer and Multifrequency EPR Study

“…33, No. 16, 1994 described (Coremans et al, 1992a). Hydrogenase activity was measured amperometrically at 30 OC and pH 8.0 with a Clark-type electrode (type YSI 5331) as described (Coremans et al, 1989).…”

“…In the present paper we have used Mossbauer and EPR spectroscopies to identify the Fe-containing centers of the C. vinosum enzyme, with the particular view of illuminating the coupling/decoupling phenomenon attending the redox process at E,(pH 8.0) = +150 mV (Coremans et al, 1992a). We will show that the enzyme contains two Fe& clusters and one Fe& cluster and we address the possibility for an additional Fe site.…”