Bardischewsky Frank, Quentmeier Armin, Rother Dagmar, Hellwig Petra, Kostka Susanne, Friedrich Cornelius G
Lehrstuhl für Technische Mikrobiologie, Fachbereich Bio- und Chemieingenieurwesen, Universität Dortmund, Emil-Figge-Strasse 66, D-44221 Dortmund, Germany.
Biochemistry. 2005 May 10;44(18):7024-34. doi: 10.1021/bi047334b.
Sulfur dehydrogenase, Sox(CD)(2), is an essential part of the sulfur-oxidizing enzyme system of the chemotrophic bacterium Paracoccus pantotrophus. Sox(CD)(2) is a alpha(2)beta(2) complex composed of the molybdoprotein SoxC (43 442 Da) and the hybrid diheme c-type cytochrome SoxD (37 637 Da). Sox(CD)(2) catalyzes the oxidation of protein-bound sulfur to sulfate with a unique six-electron transfer. Amino acid sequence analysis identified the heme-1 domain of SoxD proteins to be specific for sulfur dehydrogenases and to contain a novel ProCysMetXaaAspCys motif, while the heme-2 domain is related to various cytochromes c(2). Purification of sulfur dehydrogenase without protease inhibitor yielded a dimeric SoxCD(1) complex consisting of SoxC and SoxD(1) of 30 kDa, which contained only the heme-1 domain. The heme-2 domain was isolated as a new cytochrome SoxD(2) of about 13 kDa. Both hemes of SoxD in Sox(CD)(2) are redox-active with midpoint potentials at E(m)1 = 218 +/- 10 mV and E(m)2 = 268 +/- 10 mV, while SoxCD(1) and SoxD(2) both exhibit a midpoint potential of E(m) = 278 +/- 10 mV. Electrochemically induced FTIR difference spectra of Sox(CD)(2), SoxCD(1), and SoxD(2) were distinct. A carboxy group is protonated upon reduction of the SoxD(1) heme but not for SoxD(2). The specific activity of SoxCD(1) and Sox(CD)(2) was identical as was the yield of electrons with thiosulfate in the reconstituted Sox enzyme system. To examine the physiological significance of the heme-2 domain, a mutant was constructed that was deleted for the heme-2 domain, which produced SoxCD(1) and transferred electrons from thiosulfate to oxygen. These data demonstrated the crucial role of the heme-1 domain of SoxD for catalytic activity, electron yield, and transfer of the electrons to the cytoplasmic membrane, while the heme-2 domain mediated the alpha(2)beta(2) tetrameric structure of sulfur dehydrogenase.
硫脱氢酶Sox(CD)(2)是嗜营养副球菌化学营养细菌硫氧化酶系统的重要组成部分。Sox(CD)(2)是一种α(2)β(2)复合物，由钼蛋白SoxC（43442道尔顿）和杂合双血红素c型细胞色素SoxD（37637道尔顿）组成。Sox(CD)(2)通过独特的六电子转移催化蛋白质结合的硫氧化为硫酸盐。氨基酸序列分析表明，SoxD蛋白的血红素-1结构域对硫脱氢酶具有特异性，并且包含一个新的ProCysMetXaaAspCys基序，而血红素-2结构域与各种细胞色素c(2)相关。在没有蛋白酶抑制剂的情况下纯化硫脱氢酶，得到了由30 kDa的SoxC和SoxD(1)组成的二聚体SoxCD(1)复合物，其仅包含血红素-1结构域。血红素-2结构域被分离为约13 kDa的新细胞色素SoxD(2)。Sox(CD)(2)中SoxD的两个血红素均具有氧化还原活性，中点电位分别为E(m)1 = 218 +/- 10 mV和E(m)2 = 268 +/- 10 mV，而SoxCD(1)和SoxD(2)的中点电位均为E(m) = 278 +/- 10 mV。Sox(CD)(2)、SoxCD(1)和SoxD(2)的电化学诱导傅里叶变换红外差光谱不同。SoxD(1)血红素还原时羧基会质子化，而SoxD(2)则不会。SoxCD(1)和Sox(CD)(2)的比活性相同，并且在重组的Sox酶系统中与硫代硫酸盐的电子产率也相同。为了研究血红素-2结构域的生理意义，构建了一个缺失血红素-2结构域的突变体，该突变体产生SoxCD(1)并将电子从硫代硫酸盐转移到氧气。这些数据表明，SoxD的血红素-1结构域对于催化活性、电子产率以及电子向细胞质膜的转移起着关键作用，而血红素-2结构域介导了硫脱氢酶的α(2)β(2)四聚体结构。
