Retention of heme in axial ligand mutants of succinate-ubiquinone oxidoreductase (complex II) from Escherichia coli

Succinate-ubiquinone oxidoreductase (SdhCDAB, complex II) from Escherichia coli is a four-subunit membrane-bound respiratory complex that catalyzes ub iquinone reduction by succinate, In the E. coli enzyme, heme b(556) is liga ted between SdhC His(84) and SdhD His(71). Contrary to a previous report (V ibat, C. R. T., Cecchini, G., Nakamura, K., Kita, K,, and Gennis, R, B, (19 98) Biochemistry 37, 4148-4159), we demonstrate the presence of heme in bot h SdhC H84L and SdhD H71Q mutants of SdhCDAB. EPR spectroscopy reveals the presence of low spin heme in the SdhC H84L, (g(Z) = 2.92) mutant and high s pin heme in the SdhD H71Q mutant (g = 6.0), The presence of low spin heme i n the SdhC H84L mutant suggests that the heme b(556) is able to pick up ano ther ligand from the protein. CO binds to the reduced form of the mutants, indicating that it is able to displace one of the ligands to the low spin h eme of the SdhC H84L, mutant. The g = 2.92 signal of the SdhC H84L mutant t itrates with a redox potential at pH 7.0 (E-m,E-7) of approximately +15 mV, whereas the g 6.0 signal of the SdhD H71Q mutant titrates with an E-m,E-7 of approximately -100 mV, The quinone site inhibitor pentachlorophenol pert urbs the heme optical spectrum of the wild-type and SdhD H71Q mutant enzyme s but not the SdhC H84L mutant. This finding suggests that the latter resid ue also plays an important role in defining the quinone binding site of the enzyme. The SdhC H84L mutation also results in a significant increase in t he K, and a decrease in the k(cat) for ubiquinone-1, whereas the SdhD H71Q mutant has little effect on these parameters. Overall, these data indicate that SdhC His(84) has an important role in defining the interaction of SdhC DAB with both quinones and heme b(556).