Andreas Christenson

Direct Electrochemistry of Proteins and Enzymes

Publisher Summary This chapter summarizes the achievements on direct electron transfer (DET) between redox enzymes and electrodes, with a special focus on haem and copper-containing redox enzymes. Haem enzymes involve peroxidases, catalases, cytochromes of the P450 group, and a variety of multi-cofactor complex enzymes that contain haem(s), along with other cofactors such as flavin(s), copper, and iron–sulphur cluster(s). There are a variety of electron tunneling pathways within the enzyme molecule between the redox active centre and the protein surface. The chapter presents two groups of redox enzymes: the intrinsic and extrinsic ones. To realize efficient DET between redox enzymes and electrodes, a proper orientation of the redox enzyme onto the electrode, through the site of the electron-tunneling pathways, where the partner protein commonly binds or through the domain of the active site exposed to the protein surface, becomes important. The copper sites in the redox proteins have been divided into three classes based on their spectroscopic features that reflect the geometric and electronic structure of the active site: type 1 (T1) or blue copper, type 2 (T2) or normal copper, and type 3 (T3) or coupled binuclear copper centers. The chapter describes all copper-containing proteins that are divided into four groups according to the structure of their active sites: (1) type-1 copper proteins, (2) type-2 copper enzymes, (3) type-3 copper proteins, and (4)‘‘blue’’ multi-copper oxidases.

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.

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.