Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Abstract

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q p and Q d. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b H, E m = + 65 mV, heme b L, E m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b L, and with a pronounced delay of reoxidation. When the specific inhibitor 2- n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q d in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q p site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q p or Q d, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q p depended on the redox state of the hemes. When both hemes were reduced, and Q d was blocked by HQNO, quinone-mediated communication via the Q p site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.