Bilirubin Oxidase Adsorption onto Charged Self-Assembled Monolayers: Insights from Multiscale Simulations

Abstract

The efficient immobilization and orientation of bilirubin oxidase (BOx) on different solid substrates are essential for its application in biotechnology. The T1 copper site within BOx is responsible for the electron transfer. In order to obtain quick direct electron transfer (DET), it is important to keep the distance between the T1 copper site and electrode surface small and to maintain the natural structure of BOx at the same time. In this work, the combined parallel tempering Monte Carlo simulation with the all-atom molecular dynamics simulation approach was adopted to reveal the adsorption mechanism, orientation, and conformational changes of BOx from Myrothecium verrucaria (MvBOx) adsorbed on charged self-assembled monolayers (SAMs), including COOH-SAM and NH-SAM with different surface charge densities (±0.05 and ±0.19 C·m). The results show that MvBOx adsorbs on negatively charged surfaces with a "back-on" orientation, whereas on positively charged surfaces, MvBOx binds with a "lying-on" orientation. The locations of the T1 copper site are closer to negatively charged surfaces. Furthermore, for negatively charged surfaces, the T1 copper site prefers to orient closer to the surface with lower surface charge density. Therefore, the negatively charged surface with low surface charge density is more suitable for the DET of MvBOx on electrodes. Besides, the structural changes primarily take place on the relatively flexible turns, coils, and α-helix. The native structure of MvBOx is well preserved when it adsorbs on both charged surfaces. This work sheds light on the controlling orientation and conformational information on MvBOx on charged surfaces at the atomistic level. This understanding would certainly promote our understanding of the mechanism of MvBOx immobilization and provide theoretical support for BOx-based bioelectrode design.

Recent advances in surface chemistry of electrodes to promote direct enzymatic bioelectrocatalysis

“…New fundamental concepts driving enzyme immobilization were demonstrated: i) the global charge of the enzyme drives its loading on the interface; ii) the dipole moment of the enzyme induced by anisotropy of charges, and the environment charge around the first electron acceptor (Cu T1), drive the enzyme orientation on the surface; iii) higher enzyme coverage does not necessarily translate into higher specific activity; iv) change in local pH induces dynamics of reorientation of enzymes on electrodes. All-atom molecular dynamic simulation confirms the electrostatic model for BOD, and highlights preserved conformation of the enzyme in the immobilized state [11]. Mixed SAMs can be used to tune the number of surface functionalities and their distribution [12].…”

Electrostatic-Driven Activity, Loading, Dynamics, and Stability of a Redox Enzyme on Functionalized-Gold Electrodes for Bioelectrocatalysis

“…Understanding the factors that affect this low catalytic efficiency is thus required. While porous electrodes may enhance the loading of enzymes, planar electrodes are much more appropriate for the fundamental studies of enzyme immobilization 14,[19][20][21][22] . In particular, planar gold surfaces are mostly used in methods allowing to study loading of enzymes on solid supports (surface plasmon resonance (SPR), quartz crystal microbalance (QCM)), or enzyme conformation in the immobilized state (Surface Enhanced Infrared Absorption (SEIRA),…”

Implicit Modeling of the Impact of Adsorption on Solid Surfaces for Protein Mechanics and Activity with a Coarse-Grained Representation

“…To model the impact of protein adsorption on the rigidity profile, we selected as anchor residues the residues that were predicted to lie within 0.35 nm from the solid surface in 9 the simulation work of Yang et al 55 . In this study, the authors used a combination of parallel tempering Monte Carlo and all-atom Molecular Dynamics simulations to predict the orientation of BOD adsorbed on a SAM functionalized surface.…”

“…positively) charged surface on which the protein is adsorbed (a) (b)• Arg353, Gly358, Gly365, Asp370, Gln372 and Arg437 for the negatively charged surface. (Note that the negatively charged residue Asp370 generates a repulsion on the negatively charged SAM that is characterized and quantified in ref 55). • Asp53, Asp322, Asp323, Gln505, Ala506 and Gln507 for the positively charged surface.…”