Quentmeier Armin, Hellwig Petra, Bardischewsky Frank, Wichmann Rolf, Friedrich Cornelius G
Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany.
Biochemistry. 2004 Nov 23;43(46):14696-703. doi: 10.1021/bi048568y.
Flavocytochrome c-sulfide dehydrogenases (FCSDs) are complexes of a flavoprotein with a c-type cytochrome performing hydrogen sulfide-dependent cytochrome c reduction in vitro. The amino acid sequence analysis revealed that the phylogenetic relationship of different flavoproteins reflected the relationship of sulfur-oxidizing bacteria. The flavoprotein SoxF of Paracoccus pantotrophus is 29-67% identical to the flavoprotein subunit of FCSD of phototrophic sulfur-oxidizing bacteria. Purification of SoxF yielded a homogeneous emerald-green monomeric protein of 42 797 Da. SoxF catalyzed sulfide-dependent horse heart cytochrome c reduction at the optimum pH of 6.0 with a k(cat) of 3.9 s(-1), a K(m) of 2.3 microM for sulfide, and a K(m) of 116 microM for cytochrome c, as determined by nonlinear regression analysis. The yield of 1.9 mol of cytochrome c reduced per mole of sulfide suggests sulfur or polysulfide as the product. Sulfide dehydrogenase activity of SoxF was inhibited by sulfur (K(i) = 1.3 microM) and inactivated by sulfite. Cyanide (1 mM) inhibited SoxF activity at pH 6.0 by 25% and at pH 8.0 by 92%. Redox titrations in the infrared spectral range from 1800 to 1200 cm(-1) and in the visible spectral range from 400 to 700 nm both yielded a midpoint potential for SoxF of -555 +/- 10 mV versus Ag/AgCl at pH 7.5 and -440 +/- 20 mV versus Ag/AgCl at pH 6.0 (-232 mV versus SHE') and a transfer of 1.9 electrons. Electrochemically induced FTIR difference spectra of SoxF as compared to those of free flavin in solution suggested a strong cofactor interaction with the apoprotein. Furthermore, an activation/variation of SoxF during the redox cycles is observed. This is the first report of a monomeric flavoprotein with sulfide dehydrogenase activity.
黄素细胞色素c-硫化物脱氢酶（FCSDs）是一种黄素蛋白与c型细胞色素的复合物，在体外可进行依赖硫化氢的细胞色素c还原反应。氨基酸序列分析表明，不同黄素蛋白的系统发育关系反映了硫氧化细菌之间的关系。嗜甲基副球菌的黄素蛋白SoxF与光合硫氧化细菌FCSD的黄素蛋白亚基具有29%-67%的同一性。纯化后的SoxF得到了一种均一的、分子量为42797 Da的翠绿色单体蛋白。通过非线性回归分析测定，SoxF在最适pH 6.0时催化依赖硫化物的马心细胞色素c还原反应，催化常数k(cat)为3.9 s(-1)，对硫化物的米氏常数K(m)为2.3 μM，对细胞色素c的米氏常数K(m)为116 μM。每摩尔硫化物还原1.9摩尔细胞色素c的产率表明产物为硫或多硫化物。SoxF的硫化物脱氢酶活性受到硫的抑制（抑制常数K(i)=1.3 μM），并被亚硫酸盐灭活。氰化物（1 mM）在pH 6.0时抑制SoxF活性25%，在pH 8.0时抑制92%。在1800至1200 cm(-1)的红外光谱范围和400至700 nm的可见光谱范围内进行的氧化还原滴定，在pH 7.5时相对于Ag/AgCl测得SoxF的中点电位为-555±10 mV，在pH 6.0时相对于Ag/AgCl为-440±20 mV（相对于标准氢电极SHE'为-232 mV），电子转移数为1.9。与溶液中游离黄素的电化学诱导傅里叶变换红外差谱相比，SoxF的差谱表明辅因子与脱辅基蛋白有强烈的相互作用。此外，在氧化还原循环中观察到SoxF的激活/变化。这是关于具有硫化物脱氢酶活性的单体黄素蛋白的首次报道。
