tailieunhanh - Báo cáo khóa học: UV-A/blue-light inactivation of the ‘metal-free’ hydrogenase (Hmd) from methanogenic archaea The enzyme contains functional iron after all

H2 -forming methylenetetrahydromethanopterin dehydro-genase (Hmd) is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed metal-free hydrogenase does not contain iron–sulfur clus-ters or nickel and thus differs from [Ni-Fe] and [Fe-Fe] hydrogenases, which are all iron–sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit, but the iron was considered to be a con-taminant as none of the catalytic and spectroscopic pro-perties of the enzyme indicated that it was an essential component | Eur. J. Biochem. 271 195-204 2004 FEBS 2003 doi UV-A blue-light inactivation of the metal-free hydrogenase Hmd from methanogenic archaea The enzyme contains functional iron after all Erica J. Lvon1 . Seiao Shima1 Gerrit Buurman1 Shantanu Chowdhuri1 Alfred Batschauer2 . Klaus Steinbach3 and Rudolf K. Thauer1 1 Max-Planck-Institut fur terrestrische Mikrobiologie and 2Fachbereich Biologie der Philipps-Universitdt Marburg Germany 3Fachbereich Chemie der Philipps-Universitat Marburg Germany H2-forming methylenetetrahydromethanopterin dehydrogenase Hmd is an unusual hydrogenase present in many methanogenic archaea. The homodimeric enzyme dubbed metal-free hydrogenase does not contain iron-sulfur clusters or nickel and thus differs from Ni-Fe and Fe-Fe hydrogenases which are all iron-sulfur proteins. Hmd preparations were found to contain up to 1 mol iron per 40 kDa subunit but the iron was considered to be a contaminant as none of the catalytic and spectroscopic properties of the enzyme indicated that it was an essential component. Hmd does however harbour a low molecular mass cofactor of yet unknown structure. We report here that the iron found in Hmd is most probably functional after all. Further investigation was initiated by the discovery that Hmd is inactivated upon exposure to UV-A 320-400 nm or blue-light 400-500 nm . Enzyme purified in the dark exhibited an absorption spectrum with a maximum at approximately 360 nm and which mirrored its sensitivity towards light. In UV-A blue-light the enzyme was bleached. The cofactor extracted from active Hmd was also light sensitive. It showed an UV visible spectrum similar to that of the active enzyme and was bleached upon exposure to light. Photobleached cofactor no longer had the ability to reconstitute active Hmd from the apoenzyme. When purified in the dark Hmd consistently contained per monomer about one Fe which was tightly bound to the cofactor. The iron was released from the enzyme .