A complex heterogeneous intracellular environment seems to affect enzymatic catalysis by changing the mobility of biomolecules, their stability, and their conformational states, as well as by facilitating or hindering continuously occurring interactions. The evaluation and description of the influence of the cytoplasmic matrix components on enzymatic activity are problems that remain unsolved. In this work, we aimed to determine the mechanisms of action of two-component media with cosolvents of various molecular sizes on the complex multi-stage bioluminescent reaction catalyzed by bacterial luciferase. Kinetic and structural effects of ethylene glycol, glycerol, sorbitol, glucose, sucrose, dextran, and polyethylene glycol on bacterial luciferase were studied using stopped-flow and fluorescence spectroscopy techniques and molecular dynamics simulations. We have found that diffusion limitations in the presence of cosolvents promote the stabilization of flavin substrate and peroxyflavin intermediate of the reaction, but do not provide any advantages in bioluminescence quantum yield, because substrate binding is slowed down as well. The catalytic constant of bacterial luciferase has been found to be viscosity-independent and correlated with parameters of water-cosolvent interactions (Norrish constant, van der Waals interaction energies). Crowding agents, in contrast to low-molecular-weight cosolvents, had little effect on peroxyflavin intermediate decay and enzyme catalytic constant. We attributed specific kinetic effects to the preferential interaction of the cosolvents with enzyme surface and their penetration into the active site.

Hormesis is a biphasic dose-response relationship featured by low-dose stimulation and high-dose inhibition. Although the hormetic phenomenon has been extensively studied over the past decades, there is little information regarding the influence of energy source on the occurrence of hormesis, especially the time-dependent one. In this study, to explore the role of cultivation system\'s energy source in time-dependent hormesis, the toxic dose-responses of Aliivibrio fischeri (A. fischeri) bioluminescence to Sulfadoxine (SDX) during 24 h were determined in four cultivation systems with different energy source conditions. The results indicated that the time-dependent hormetic effects were induced by SDX in all cultivation systems: SDX triggered hormetic phenomenon on the bioluminescence at each growth stage over 24 h in the cultivation systems with sufficient and insufficient energy source; due to the diauxic growth of A. fischeri under multiple energy source conditions, the hormetic effects of SDX gradually disappeared after the preferred energy source was used up. It was speculated that the inhibitory action of SDX was derived from its interaction with DHPS to impede the synthesis of proteins, and SDX bound with AC to upregulate the quorum sensing (QS) system to exhibit the stimulatory action. Comparing the time-dependent hormesis in each cultivation system, it was obtained that the energy source could impact the hourly maximum stimulatory rate, the EC50 of SDX, and the time point that hormesis occurred, which might result from the influence of energy source on the stimulatory and inhibitory actions of SDX through regulating the metabolic system (individual level) and QS system (group level) of bacteria. This study clarifies the importance of energy source for hormesis occurrence, which may further promote the development of hormesis.

In less than half a century, the hybrid QM/MM method has become one of the most used technique to model molecules embedded in a complex environment. A well-known application of the QM/MM method is for biological systems. Nowadays, one can understand how enzymatic reactions work or compute spectroscopic properties, like the wavelength of emission. Here, we have tackled the issue of modeling chemical reactions inside proteins. We have studied a bioluminescent system, fireflies, and deciphered if a keto-enol tautomerization is possible inside the protein. The two tautomers are candidates to be the emissive molecule of the bioluminescence but no outcome has been reached. One hypothesis is to consider a possible keto-enol tautomerization to treat this issue, as it has been already observed in water. A joint approach combining extensive MD simulations as well as computation of key intermediates like TS using QM/MM calculations is presented in this publication. We also emphasize the procedure and difficulties met during this approach in order to give a guide for this kind of chemical reactions using QM/MM methods.