Immobilization of proteins onto solid supports has critical industrial, technological, and medical applications, and is a daily task in chemical research. Significant conformational rearrangements often occur due to enzyme–surface interactions, and it is of broad interest to develop methods to probe and better understand these molecular-level changes that contribute to the enzyme’s catalytic activity and stability. While circular dichroism is a common method for solution-phase conformational study, the application to surface-supported proteins is not trivial and spatial mapping is not viable. On the other hand, a nonlinear laser spectroscopy technique used to analyze surfaces and interfaces is not often found in most laboratories, therefore requiring an alternative and reliable method. Here, we employed high-dimensional data spectromicroscopy analysis in the infrared region (μ-FTIR) to investigate the enzyme’s conformational change when adsorbed onto solid matrices, across a ca. 20 mm2 area. Alcohol dehydrogenase (ADH) enzyme was adopted as a model enzyme to interact with CaF2, Au, and Au-thiol model substrates, strategically chosen for mapping the enzyme dynamics on solid surfaces with different polarity/hydrophobicity properties and extendable to other materials. Two-dimensional chemical maps indicate that the enzyme adsorbs with different patterns in which secondary structures dynamically adjust to optimize interprotein and enzyme–surface interactions. The results suggest an experimental approach to identify and map enzyme conformational dynamics onto different solid surfaces across space and provide insights into immobilized protein structure investigations for areas such as biosensing and bioenergy.

Synthetic biology pursues the understanding of biological processes in a molecular level, as well as, the design of bioinspired materials, with analogous or improved properties as compared to the living organisms, able to mimicry those processes. A number of materials have already been studied and applied as redox proteins mimetics and, among these, iron oxides nanoparticles have shown themselves as potential candidates in the mimicry of processes involving, mainly, electron transfer reactions. In this study, we propose the mimicry of an important redox step of the mitochondrial respiratory chain, applying iron oxides-based nanomaterials and a heme protein, the cytochrome c. For such, magnetite nanoparticles (Fe3O4) and maghemite nanoparticles (-Fe2O3) were synthesized and the nanoparticle-protein interaction was, initially, proven in solution and, further, in a mitochondrial membrane model (Langmuir-Blodgett film). In situ experiments showed interactions highly favored by coulombic forces and van der Waals, resulting in a direct electron transfer between the superficial iron sites of the nanoparticles and the protein heme group, which by consequence changes the oxidation state of this prosthetic group, but not affecting the protein secondary and tertiary structures, though. We observed that the Fe3O4 nanoparticles behave as a complex III (cytochrome bc1) redox mimetic, reducing the ferricytochrome c (Fe 3+-heme) to ferrouscytochrome c (Fe 2+-heme) with a second-order heterogeneous electron transfer rate constant of 2.63 ± 0.05 L mol-1 s-1 at 25ºC, with an activation energy of 40.2 ± 1.5 kJ mol-1. The-Fe2O3 nanoparticles, on the other hand, act as a complex IV (cytochrome c oxidase) redox mimetic, re-oxidizing ferrouscytochrome c to ferricytochrome c. The results indicated that the extent of the reaction depends on the Fe 2+ /Fe 3+ ratio on the surface of the nanoparticles, as superficial Fe 2+ ions control the ferricytochrome c reduction, while ferrouscytochrome c oxidation depends on superficial Fe 3+ ions. We also observed that, when incorporated in a mitochondrial cell membrane model, both nanoparticles maintain their redox activity towards cytochrome c, indicating that inorganic nanostructured systems may behave as protein complexes in a cellular membrane model.