Nitric Oxide Synthase Oxygenase in Thin Layer-By-Layer Polymeric Films: Does the pH at Which the Film Is Built Affect the Direct Electron Transfer or the Related NOS-Mediated Catalysis?
Authors: Bayachou Mekki , Gunesekera Bhagya , Kalil Haitham , Maher Shaimaa a. , Abou Diwan Charbel 
Published: May 2020

We prepared thin films containing nitric oxide synthase (NOS) oxygenase enzymes and polyethyleneimine (PEI) using the layer-by-layer film building strategy. The charge-based layer-by-layer electrostatic adsorption allows for assembly of multi-component protein/PEI films. During the film building process, we used NOS oxygenase enzyme from solutions at two distinct pHs (pH=7.0 and pH=8.6) to study the effect of pH on enzyme loading in the thin polymeric film. We studied direct electron transfer to NOS oxygenase in the two kinds of films at physiologic pH. We show that NOS oxygenase enzymes maintain their overall integral structure in PEI films in both pH conditions, and exhibit relatively fast but similar electron transfer rates. Using thin film coulometry, we show that the enzyme loading from pH 8.6 is almost 4-times more than the amount loaded in films prepared using NOS oxygenase solutions at pH=7.0.
To closely examine the effect of pH during the layer-by-layer film building on the trapped NOS oxygenase enzymes, we studied the catalytic reduction of exogenous nitric oxide analyte mediated by NOS oxygenases in PEI films. We used this property to compare and contrast the catalytic activity of the films of NOS enzymes prepared at pH 7.0 and those prepared at pH 8.6. Our results show that there is a distinct increase in catalytic activity of enzymes adsorbed from pH 8.6 compared to those prepared at pH=7.0. We also show that the measured differential loading amnounts in the two kinds of films cannot explain the observed increase in mediated catalytic activity. In this regard, we show that NOS oxygenase films constructed at pH 8.6 exhibit significantly higher catalytic efficiency (kcat
/KM) compared to films constructed at pH 7.0. To better understand the change in catalytic activity, we used detailed Fourier Transform Infrared (FTIR) spectroscopic analysis of the thin films to closely examine the structural integrity of the trapped NOS oxygenases in the two sets of films. Deconvolution methods using FTIR data suggest that although NOS oxygenase enzymes maintain their overall structure, we observe distinct pH-dependent conformational changes, which includes a decrease in α-helix contributions in the NOS protein adsorbed at pH 8.6. The observed changes in the secondary structure apparently add to the disordering of protective loops that affect a hydrophobic pocket trap that is critical for NO binding during catalysis.This finding is in line with the observed change in NO affinity for the trapped NOS enzymes as evidenced by the distinct change in catalytic efficiency of NOS oxygenases in the two sets of films. This work shows that while the electron transfer to NOS enzymes in PEI films is not affected by pH in which the layer-by-layer film fabrication occurs, the subtle pH-induced backbone changes provides a structural basis for the higher specific catalytic electrochemical reduction of NO by NOS oxygenases in PEI films constructed at pH 8.6.