Control of carbon monoxide dehydrogenase orientation by site-specific immobilization enables direct electrical contact between enzyme cofactor and solid surface

Abstract

Controlling the orientation of redox enzymes on electrode surfaces is essential in the development of direct electron transfer (DET)-based bioelectrocatalytic systems. The electron transfer (ET) distance varies according to the enzyme orientation when immobilized on an electrode surface, which influences the interfacial ET rate. We report control of the orientation of carbon monoxide dehydrogenase (CODH) as a model enzyme through the fusion of gold-binding peptide (gbp) at either the N- or the C-terminus, and at both termini to strengthen the binding interactions between the fusion enzyme and the gold surface. Key factors influenced by the gbp fusion site are described. Collectively, our data show that control of the CODH orientation on an electrode surface is achieved through the presence of dual tethering sites, which maintains the enzyme cofactor within a DET-available distance (<14 Å), thereby promoting DET at the enzyme–electrode interface.

Electrochemical transformations catalyzed by cytochrome P450s and peroxidases

“…Therefore, the configuration of heme enzymes in a monolayer, for example, needs to be orientated to minimize the distance between the heme center and electrode (within 14 Å) as a so-called ''electroactive'' configuration. [209][210][211] HRP, for example, has a molecular radius of 30 Å 212 that would block the direct electron transfer, since the heme sites buried in the protein frameworks may be too far away from the electrode. Previous literature suggested that the selectivity and stability of enzymes can also be controlled by varying the orientation of enzymes on the electrode surface.…”

“…Adding a short electrode-binding peptide at a specific terminal of enzymes can guide the binding direction of enzymes to the electrode and avoid the non-specifical orientation of enzymes. 209 This method can provide the oriented immobilization of Cyt P450s. 234 P450 BM3 C-terminally high specific peptide (HSP), as an example can be immobilized on an ITO electrode binding peptide (HSP) made of sequence (RTRHK) 4 .…”

Versatile Biosynthetic Approach to Regioselective Enzyme Patterning for Direct Bioelectrocatalysis Application

“…Effects of design factors on performance parameters References SBP fusion site Catalytic activity ✓ Catalytic activity of enzyme tends to be highly preserved after genetic SBP fusion. [29,34,44] Inorganic-binding activity ✓ SBP fusion to multiple sites can increase overa ll inorganic-binding affinities. ✓ The external exposure degree of fusion site (i.e., C-or N-terminus of protein structure) can determine contact probability of SBP domain with target surfaces.…”

“…[ 27 ] Therefore, to achieve intimate, direct electrical coupling between redox enzymes and electrodes, the enzyme must be oriented on the electrode such that its active site is near the metallic surface. [ 28,29 ]…”

“…[27] Therefore, to achieve intimate, direct electrical coupling between redox enzymes and electrodes, the enzyme must be oriented on the electrode such that its active site is near the metallic surface. [28,29] Taken together, the immobilization strategy for enzyme patterning of the enzyme-based direct bioelectrocatalytic system should also control the electrical conductivity of redox cofactors with the electrode surface while maintaining the catalytic activity of the native enzymes in a DET-based enzyme-electrode system. In other words, "multi-faceted control" of cofactor-tosurface orientation and binding position of the enzyme on the electrode should be accomplished.…”