Kowal A T, Werth M T, Manodori A, Cecchini G, Schröder I, Gunsalus R P, Johnson M K
Department of Chemistry, University of Georgia, Athens 30602, USA.
Biochemistry. 1995 Sep 26;34(38):12284-93. doi: 10.1021/bi00038a024.
Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154, and Cys158 are replaced individually by Ser have been constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters have been investigated. The Cys154Ser and Cys158Ser FrdB mutations result in enzymes with negligible activity that have largely dissociated from the cytoplasmic membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters. In contrast the Cys148Ser and Cys151Ser FrdB mutations result in functional membrane bound enzymes that are able to support growth under anaerobic and aerobic conditions. EPR studies of these mutants indicate that all three of the constituent Fe-S clusters are assembled, and the redox and spectroscopic properties of the [2Fe-2S] and [3Fe-4S] clusters are unchanged compared to the wild-type enzyme. In both mutants the [4Fe-4S] cluster is assembled with one non-cysteinyl ligand, and the available data suggest serinate coordination. The physicochemical consequences are perturbation of the intercluster spin interaction between the S = 1/2 [4Fe-4S]+ and S = 2 [3Fe-FS]0 clusters and a 60-mV decrease in redox potential for the [4Fe-FS]2+,+ cluster in the FrdB Cys148Ser mutant, and a S = 1/2 to S = 3/2 spin state conversion for the [4Fe-4S]+ cluster and a 72-mV decrease in redox potential for the [4Fe-4S]2+,+ cluster in the FrdB Cys151Ser mutant. Taken together with the previous FrdB Cys to Ser mutagenesis results [Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965-8969; Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703-2712], the results provide strong support for the proposal that all three clusters are located in the FrdB subunit with Cys57, Cys62, Cys65, and Cys77 ligating the [2Fe-2S] cluster, Cys148, Cys151, Cys154, and Cys214 ligating the [4Fe-4S] cluster, and Cys158, Cys204, and Cys210 ligating the [3Fe-4S] cluster. The role of the low potential [4Fe-4S] cluster in mediating electron transfer from menaquinol to the FAD active site is discussed in light of these mutagenesis results.
已构建了大肠杆菌延胡索酸还原酶的定点突变体，其中FrdB的半胱氨酸148、半胱氨酸151、半胱氨酸154和半胱氨酸158分别被丝氨酸取代，并在缺乏野生型延胡索酸还原酶和琥珀酸脱氢酶的大肠杆菌菌株中进行了过表达。研究了这些突变对细菌生长、酶活性以及组成型铁硫簇的电子顺磁共振（EPR）特性的影响。半胱氨酸154丝氨酸和半胱氨酸158丝氨酸的FrdB突变导致酶活性可忽略不计，这些酶已基本从细胞质膜解离，因此在需要功能性延胡索酸还原酶的条件下无法支持细胞生长。EPR研究表明，这些影响与[3Fe-4S]和[4Fe-4S]簇的丢失有关。相比之下，半胱氨酸148丝氨酸和半胱氨酸151丝氨酸的FrdB突变产生了功能性膜结合酶，能够在厌氧和好氧条件下支持生长。对这些突变体的EPR研究表明，所有三个组成型铁硫簇都已组装，并且与野生型酶相比，[2Fe-2S]和[3Fe-4S]簇的氧化还原和光谱特性没有变化。在这两个突变体中，[4Fe-4S]簇都与一个非半胱氨酸配体组装，现有数据表明是丝氨酸盐配位。其物理化学结果是，在FrdB半胱氨酸148丝氨酸突变体中，S = 1/2的[4Fe-4S]+和S = 2的[3Fe-FS]0簇之间的簇间自旋相互作用受到扰动，[4Fe-FS]2+,+簇的氧化还原电位降低60 mV；在FrdB半胱氨酸151丝氨酸突变体中，[4Fe-4S]+簇发生S = 1/2到S = 3/2的自旋态转换，[4Fe-4S]2+,+簇的氧化还原电位降低72 mV。结合之前FrdB半胱氨酸到丝氨酸的诱变结果[Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965 - 8969; Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703 - 2712]，这些结果为以下提议提供了有力支持：所有三个簇都位于FrdB亚基中，半胱氨酸57、半胱氨酸62、半胱氨酸65和半胱氨酸77连接[2Fe-2S]簇，半胱氨酸148、半胱氨酸151、半胱氨酸154和半胱氨酸214连接[4Fe-4S]簇，半胱氨酸158、半胱氨酸204和半胱氨酸210连接[3Fe-4S]簇。根据这些诱变结果，讨论了低电位[4Fe-4S]簇在介导从甲基萘醌到FAD活性位点的电子转移中的作用。
