Electron transfer dynamics in iron-sulfur proteins: structural and environmental factors controlling efficiency

Electron transfer (ET) is a fundamental process in biology, being important in biological energy conversion and catalysis, enabling diverse redox transformations in living systems. Understanding the molecular drivers of ET in iron-sulfur (Fe-S) cluster proteins and flavoprotein complexes both deepens insights into natural redox networks and informs the design of next-generation biomedical and bioelectrocatalytic devices. This Ph.D. research integrates advanced computational methods (coarse-grained and atomistic molecular dynamics, docking, mediator mapping) with experimental strategies to study ET mechanisms at a molecular level. Particular emphasis has been placed on two protein systems of applied and biological relevance: the formate dehydrogenase (FDH)–membrane-bound hydrogenase (MBH) complex and the flavoprotein redox interaction between human SAND (hSAND) and cytochrome b5 reductase (CYB5R) isoforms. Key findings are as follows: Chapter 4 overall structural stability and region-specific dynamics of Formate dehydrogenase from Rhodobacter capsulatus (RcFDH) and membrane-bound hydrogenase from Cupriavidus necator (CnMBH) from coarse-grained simulations. Chapter 5 combines docking and CG simulations of RcFDH-CnMBH complex in solution with experimental validation. The results show complexes are weakly bound and cofactors are positioned unfavorably for direct ET. Only one configuration supports efficient tunneling. In situ IR spectroscopy and enzymatic assays confirm that ET is intrinsically inefficient in the absence of mediators. Methyl viologen (MV) enhances coupling consistent with computed binding hotspots near FMN and the A4 in the FDH, and in the case of the hydrogenase at the distal 4Fe-4S cofactor. Chapter 6 explores immobilization of RcFDH and CnMBH on self-assembled monolayer (SAM) surfaces. On SAM electrodes, surface chemistry and ionic strength set adsorption and cofactor–electrode distance. COOH SAMs at 50 mM fix orientation but keep cofactors beyond tunneling range. Whereas mixed and amine-terminated SAMs broaden orientations, only a few are DET compatible. Co-immobilization usually increases the inter-enzyme distance compared to the solution, so direct ET drops; mediators or interface redesign are needed. Chapter 7 examines electron transfer between hSAND and multiple CYB5R isoforms, showing that efficient transfer requires not only close redox‑center proximity but also a favorable local residue/electrostatic environment, with CYB5R3 emerging as the preferred partner. Mutational analysis around the hSAND FMN confirms that small microenvironmental changes can markedly alter ET rates without altering complex stability. Efficient ET requires more than short distances. Orientation, local microenvironment, and sometimes electron mediators, positioning altogether set the rate. Together, computational and experimental results define design rules for engineering enzyme interfaces and bioelectrodes for energy conversion and biosensing. While the RcFDH–CnMBH system offers clear applied potential in bioelectrocatalysis, the hSAND–CYB5R system provides mechanistic insights into ET specificity that can guide future bioengineering and biomedical strategies.