646

Abstracts

M13

THE COENZYME F420-REACTIVE HYDROGENASE OF M E -

THANOSARCINA
B A R K E R I IS A (NIFE)-ENZYME WITH F A D
AS THE PROSTHETIC GROUP
R. Michel a, C. Massanz a, M. Miiller a, M. Richter b and K. Fiebi2 a

a Institutfiir Pflanzenphysiologie und Mikrobiologie der FU Berlin, K6nigin-Luise-Str.

12-16a, D-14195 Berlin, Germany; b Institat fiir Anorganische Chemie I der Universitiit
Bielefeld, Universitiitsstr. 25, D-33615 Bielefeld, Germany

The coenzyme F420-reactive hydrogenase (FRH) from the methanogenic archaeon Methanosarina (Ms.) barked is a membrane-associated (NiFe)-protein with FAD as a prosthetic group [ 1-3]. The native hydrogenase exists in
two forms with apparent molecular masses of approx. 845 kDa and 198
kDa. The predominant 198-kDa species is composed of three non-identical
subunits with apparent molecular masses of 48 kDa (u), 33 kDa (13) and 30
kDa (y), apparently in a stoichiometry of tx2~y 1' This minimal FRH forms
macromolecular aggregates of approx. 845 kDa. One mol of 198-kDa enzyme contained 2 mol of FAD, 2 tool of nickel, 28 to 32 tool of non-heme
iron and 34 mol of acid-labile sulfur; in addition 0.2 mol of selenium were
detected. On the basis of the nucleotide sequences of the genes encoding the
three known hydrogenase polypeptides, a preliminary assignment of metals
and cofactor to the individual peptides will be proposed.
The K m of the purified FRH for H 2 with coenzyme F420 as electron
acceptor was extremely low, namely 3 ~M, and the corresponding catalytic
efficiency coefficient (kcat/Km) was high, namely 2.5 ' 107 M -1 s -1, indicating that the enzyme operates close to diffusion-limited efficiency. The amino
acid sequence PX2RXEGH, where X is any amino acid, was found to be
conserved in the N-termini of the putative nickel-binding subunits of most
(NiFe)- and (NiFeSe)-hydrogenases of methanogenic Archaea and Bacteria,
but not in the corresponding region of the (Fe)-hydrogenases. A possible
function of this motif in metal coordination [4] will be discussed.
1. K. Fiebig and B. Friedrich, Eur. J. Biochem. 184, 79 (1989)
2. H. Liinsdorf, M. Niedrig and K. Fiebig, J. Bacteriol., 173", 978 (1991)
3. R. Michel, C. Massanz, M. Richter, S. Kostka and K. Fiebig, Eur. J. Biochem. submitted
4. A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey and J. C. FontecillaCamps, Nature, 3 7 3,580 (1995)