UV-A/blue-light inactivation of the 'metal-free' hydrogenase (Hmd) from methanogenic archaea.
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 and from the cofactor upon light inactivation. Hmd activity was inhibited by high concentrations of CO and CO protected the enzyme from light inactivation indicating that the iron in Hmd is of functional importance. Therefore, reference to Hmd as 'metal-free' hydrogenase is no longer appropriate.
Lyon
Erica J
EJ
Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany.
Shima
Seigo
S
Buurman
Gerrit
G
Chowdhuri
Shantanu
S
Batschauer
Alfred
A
Steinbach
Klaus
K
Thauer
Rudolf K
RK
D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000528:radiation effects; D002850:Chromatography, Gel; D006864:Hydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000528:radiation effects*; D007501:Iron / Q000032:analysis; D007700:Kinetics; D008027:Light; D017013:Methanobacteriaceae / Q000201:enzymology*; D017014:Methanobacterium / Q000201:enzymology*; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D019843:Archaeal Proteins; D007501:Iron; D006864:Hydrogenase