PURIFICATION AND PROPERTIES OF THE HEME-CONTAINING AND IRON-SULFUR-CONTAINING HETERODISULFIDE REDUCTASE FROM METHANOSARCINA-THERMOPHILA

The heterodisulfide reductase (HDR) from Methanosarcina thermophila wa s purified to homogeneity from acetate-grown cells. In the absence of detergents, HDR consisted of an eight-protein complex with hydrogenase activity. However, when HDR was purified in the presence of 0.6% Trit on X-100, a two-subunit (53 and 27 kDa) high specific activity (simila r to 200 units mg(-1)) enzyme was obtained that lacked hydrogenase act ivity. Sedimentation equilibrium experiments demonstrated that HDR has a molecular mass of 206 kDa and a high partial specific volume (0.9 c m(3)/g), indicating that the purified protein contains a significant a mount of bound lipid. Like the HDR from Methanosarcina barkeri [Kunkel , A., Vaupel, M., Helm, S., Thauer, R. K., and Hedderich, R. (1997) Eu r. J. Biochem. 244, 226-234], it was found to contain two discrete b-t ype hemes in the small subunit and two distinct [Fe4S4](2+/1+) cluster s in the large subunit. One heme is high-spin and has a high midpoint potential (-23 mV), whereas the other heme apparently is low-spin and exhibits a relatively low midpoint potential (-180 mV). Only the high- spin heme binds CO. The midpoint potentials for the two clusters are - 100 and -400 mV. In the fully reduced state, a complicated EPR spectru m with g values of 2.03, 1.97, 1.92, and 1.88 was observed. This spect rum resembles that of 8Fe ferredoxins in the fully reduced state, indi cating that the two clusters in HDR are near enough to experience rela tively strong dipolar interactions. Kinetic studies in which CO oxidat ion is coupled to heterodisulfide reduction strongly indicate that a m embrane-associated compound is the direct electron donor to HDR. An el ectron-transfer pathway is presented that postulates a mechanism for c oupling electron transport to proton translocation.