Seedorf Henning, Dreisbach Annette, Hedderich Reiner, Shima Seigo, Thauer Rudolf K
Max-Planck-Institut für terrestrische Mikrobiologie und Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, 35043 Marburg, Germany.
Arch Microbiol. 2004 Oct;182(2-3):126-37. doi: 10.1007/s00203-004-0675-3. Epub 2004 Aug 31.
Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O(2) with H(2) at a maximal specific rate of 0.4 U (micromol/min) per mg protein with an apparent K(m) for O(2) of 30 microM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F(420)-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F(420)H(2) with 1 O(2) to 2 F(420) and 2 H(2)O. The apparent K(m) for F(420) was 30 microM and that for O(2) was 2 microM with a V(max) of 240 U/mg at 37 degrees C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H(2)O(2) as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H-X-E-X-D-X(62)-H-X(18)-D-X(60)-H. Sequence comparisons revealed that the F(420)H(2) oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30-40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F(420)H(2) oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F(420)H(2) oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.
嗜树栖甲烷短杆菌的细胞悬液催化了氢气对氧气的还原反应，其最大比速率为每毫克蛋白质0.4 U（微摩尔/分钟），氧气的表观米氏常数（K(m)）为30微摩尔。该反应不受氰化物抑制。氧化酶活性可追溯到一种依赖辅酶F(420)的酶，该酶被纯化至表观均一性，催化2分子F(420)H(2)与1分子氧气反应生成2分子F(420)和2分子水。F(420)的表观米氏常数为30微摩尔，氧气的表观米氏常数为2微摩尔，在37℃和pH 7.6（氧化酶的最适pH）条件下，最大反应速率（V(max)）为240 U/mg。该酶不使用NADH或NADPH作为电子供体，也不使用过氧化氢作为电子受体，且不受氰化物抑制。已克隆并测序了该45 kDa蛋白质的基因，每摩尔该蛋白质含有1分子FMN，并含有一个双核铁中心，序列基序为H-X-E-X-D-X(62)-H-X(18)-D-X(60)-H。序列比较表明，嗜树栖甲烷短杆菌的F(420)H(2)氧化酶在系统发育上与马尔堡嗜热栖甲烷杆菌的FprA密切相关（序列同一性为71%），FprA是一种迄今功能未知的45 kDa黄素蛋白，与细菌的A型黄素蛋白也有30%-40%的相关性，它们都具有双加氧还原酶活性。通过异源表达马尔堡嗜热栖甲烷杆菌的FprA表明，该蛋白质也是一种高效的F(420)H(2)氧化酶，且含有1分子FMN和2个铁原子。产甲烷古菌中存在F(420)H(2)氧化酶，这或许可以解释为什么一些产甲烷菌，比如白蚁后肠中的甲烷短杆菌属物种，不仅能够耐受微氧条件，还能在其中茁壮成长。
