The hyperthermophilic bacterium, Thermotoga maritima, contains an unusually complex iron-hydrogenase: amino acid sequence analyses versus biochemical characterization.
The hyperthermophilic bacterium, Thermotoga maritima, grows up to 90 degrees C by fermenting carbohydrates and it disposes of excess reductant by H(2) production. The H(2)-evolving cytoplasmic hydrogenase of this organism was shown to consist of three different subunits of masses 73 (alpha), 68 (beta) and 19 (gamma) kDa and to contain iron as the only metal. The genes encoding the subunits were clustered in a single operon in the order hydC (gamma), hydB (beta), and hydA (alpha). Sequence analyses indicated that: (a) the enzyme is an Fe-S-cluster-containing flavoprotein which uses NADH as an electron donor; and (b) the catalytic Fe-S cluster resides within the alpha-subunit, which is equivalent to the single subunit that constitutes most mesophilic Fe-hydrogenases. The alpha- and beta-subunits of the purified enzyme were separated by chromatography in the presence of 4 M urea. As predicted, the H(2)-dependent methyl viologen reduction activity of the holoenzyme (45-70 U mg(-1)) was retained in the alpha-subunit (130-160 U mg(-1)) after subunit separation. However, the holoenzyme did not contain flavin and neither it nor the alpha-subunit used NAD(P)(H) or T. maritima ferredoxin as an electron carrier. The holoenzyme, but not the alpha-subunit, reduced anthraquinone-2,6-disulfonate (apparent K(m), 690 microM) with H(2). The EPR properties of the reduced holoenzyme, when compared with those of the separated and reduced subunits, indicate the presence of a catalytic 'H-cluster' and three [4Fe-4S] and one [2Fe-2S] cluster in the alpha-subunit, together with one [4Fe-4S] and two [2Fe-2S] clusters in the beta-subunit. Sequence analyses predict that the alpha-subunit should contain an additional [2Fe-2S] cluster, while the beta-subunit should contain one [2Fe-2S] and three [4Fe-4S] clusters. The latter cluster contents are consistent with the measured Fe contents of about 32, 20 and 14 Fe mol(-1) for the holoenzyme and the alpha- and beta-subunits, respectively. The T. maritima enzyme is the first 'complex' Fe-hydrogenase to be purified and characterized, although the reason for its complexity remains unclear.
Verhagen
M F
MF
Department of Biochemistry and Molecular Biology and the Center for Metalloenzyme Studies, Life Sciences Building, The University of Georgia, Athens, GA, USA.
O'Rourke
T
T
Adams
M W
MW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C118830:iron hydrogenase; D006864:Hydrogenase