**4. Conclusions and Perspectives**

Redox proteins and enzymes immobilized on solid surfaces are fragile biomolecular entities. With a few exceptions, they retain ET of enzyme function only in prepared microenvironments that somehow emulate their natural aqueous/membrane reaction media. Self-assembled molecular monolayers with multifariously functionalized thiols are close to ideal SAM building blocks which have emerged as the core class over the last couple of decades. The -SH thiol end ascertains robust Au-S linking which is now increasingly well understood as dominated by the Au(0)-•S(0) gold-thiyl and strong van der Waals Au-S interaction. The opposite functionalized end of the SAM thiol molecules offers the choice of hydrophilic or hydrophobic, electrostatically charged or neutral, and structurally large or small terminal groups, well suited for designed gentle protein/enzyme linking to the solid

SAM-modified electrochemical surface. The option of varying the length of the SAM-forming molecules offers additional control of the electron tunneling process as well as of the dielectric and other local environmental properties crucial in the overall control of the electrochemical activity of the immobilized proteins or enzymes.

We have first overviewed the preparation and comprehensive characterization of thiol-based SAMs on Au surfaces in particular, both *per se* and as developed in redox protein/enzyme electrochemical research over the last couple of decades. Preparation is, in principle, straightforward, although the ultimate SAM properties depend in subtle ways on thiol exposure time, temperature, and other external controlling factors. A variety of sophisticated SAM surface techniques have brought the understanding of the fundamental molecular and electronic SAM structure to a high level, ranging all the way from the ordered domain right down to the single molecule. The techniques include spectroscopy (XPS, FTIR, SPR, Raman, NEXUS, and others), microscopy (AFM, STM in the electrochemical in situ/*operando* modes), and mass balance techniques (QCM), supported by large-scale electronic structure calculations [4,6,16,17,66].

Well-defined solid SAM-modified Au- and other electrode surfaces are a pre-requisite for the productive immobilization of bioelectrochemically active DET enzymes. An outstanding challenge is that polycrystalline Au- and other metallic electrodes are nearly always used, with single-crystal, atomically planar, e.g. Au(111), electrodes only relatively recently introduced as electrochemical biomolecular target surfaces. A variety of local low- and higher-index surface structures are distributed over the polycrystalline Au- and other surfaces, expectedly giving quite different surface ET activities [92]. This presents a challenge to the microscopic characterization of the pure and SAM-modified electrode surfaces but at the same time also provides new openings in the way of more robust protein/enzyme monolayers and higher enzyme activity, if the polycrystallinity can be controlled such as for NPG and other nanoporous metallic electrodes.

We have next overviewed and discussed adsorption and controlled protein and enzyme orientation on strategically chosen SAMs, of both simple ET metalloproteins (cyt. *c* and *c*4, azurin) and a variety of much more complex redox metallo- and nonmetalloenzymes (blue copper oxidases, FAD-based enzymes, cellobiose dehydrogenase, [FeS]-cluster dehydrogenases). We have shown that properly chosen SAMs can be brought to control efficiently the enzyme surface orientation in the ways most favorable both for productive bioelectrocatalysis and for detailed mapping of the molecular mechanisms involved. We have also shown that, although of more complex molecular/atomic surface structure, SAM-modified NPG and other nanoporous metallic electrochemical surfaces, structurally characterized to intermediate levels of resolution, may offer other advantages. These extend to increased enzyme stability and even enhanced catalytic efficiency compared to atomically planar electrode surfaces.

Overall, the present state of detailed structural and mechanistic protein and enzyme bioelectrochemical mapping has now advanced in impressive detail, approaching the true level of the single molecule [3,4,6,168,169] and supported by large-scale electronic structure computations [16,17,92,138]. With new and rapidly increasing understanding of the complex, heterogeneous, and anisotropic electrode/SAM/protein/enzyme/aqueous interface, its real exploitation in the strategic design and development of enzyme biofuel cells, next-generation bioelectrochemical sensors, and other high-technology applications is rapidly coming close.

**Author Contributions:** Writing—original draft, X.Y.; Supervision, D.T., J.U., X.X.; Writing—review and editing, J.T., D.T., J.U., X.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 713683. Financial support was received also from the Danish Council for Independent Research for the YDUN project (DFF 4093-00297), the Russian Science Foundation (project No. 17-13-01274), and from Villum Experiment (grant No. 35844).

**Acknowledgments:** X.Y. acknowledges support from the China Scholarship Council (No. 201806650009).

**Conflicts of Interest:** The authors declare no conflict of interest.
