Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redox-active centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.

硫化物的氧化作用是由古老的膜结合硫化物：醌氧化还原酶（SQR）催化的，该酶分为六种不同类型。为了催化硫化物的氧化，所有SQR都需要FAD辅因子和活性位点上的氧化还原活性中心，该中心通常在保守的必需半胱氨酸之间形成。不同类型的SQR在半胱氨酸的数量和位置上都有差异，这突出了各种催化机制的潜力。光合紫色硫细菌Thiocapsa roseopersicina含有具有异常催化参数的VI型SQR酶（TrSqrF）和可能参与催化作用的四个半胱氨酸。应用定点诱变来确定半胱氨酸在TrSqrF催化过程中的作用。基于这些TrSqrF变体的生化和动力学特征，Cys121被认为对酶活性至关重要。辅助因子通过Cys121和FAD的C8M基团之间的杂二硫键共价结合。所有SQR中存在的另一个半胱氨酸突变（Cys332）会导致酶活性显着降低（野生型酶的14.6％），这在酶催化中具有重要但非必需的作用。巯基阻滞剂碘乙酰胺可不可逆地灭活TrSqrF，但前提是存在底物且酶在积极催化其反应。当该酶被碘乙酰胺抑制时，FAD辅因子被释放。抑制研究支持了一种机制，该机制要求在TrSqrF的催化循环中打开和重整杂二硫键。因此，我们的研究报告了VI SQR型酶的第一个详细的结构-功能分析，该分析使这一类硫化物氧化的独特机理得以提出。