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 |
| |
Authors: | Andreas Christenson Tobias Gustavsson Lo Gorton |
| |
Institution: | a Department of Analytical Chemistry, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden b Department of Biochemistry, Lund University, P. O. Box 124, SE-221 00 Lund, Sweden |
| |
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, Qp and Qd. 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 bH, Em = + 65 mV, heme bL, Em = − 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 bL, and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Qd 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 Qp 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 Qp or Qd, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Qp depended on the redox state of the hemes. When both hemes were reduced, and Qd was blocked by HQNO, quinone-mediated communication via the Qp site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain. |
| |
Keywords: | SQR succinate:quinone oxidoreductase CV cyclic voltammetry SCE saturated calomel electrode DET direct electron transfer MET mediated electron transfer redox reduction-oxidation NQ 1 4-naphthoquinone DMN 2 3-dimethyl-1 4-naphthoquinone HQNO 2-n-heptyl-4 hydroxyquinoline N-oxide DDM dodecylmaltoside PMS phenazine methosulfate DCPIP 2 6-dichlorophenolindophenol |
本文献已被 ScienceDirect 等数据库收录! |
|