Thermodynamic and spectroscopic investigations of Shewanella putrefaciens CN32 cell envelope

Abstract

A quantitative understanding of prokaryotic cell envelope reactivity is necessary to accurately describe the surface complexation reactions known to impact metal transport in the subsurface. The gram-negative bacterium Shewanella putrefaciens CN32 (CN32) has been the focus of previous cell envelope studies in large part due to its ability to directly influence iron and uranium geochemistry; however, important gaps remain in our understanding of reactions occurring on its cell envelope. Combining surface complexation modeling of potentiometric titration data with isothermal titration calorimetry provides an additional method to evaluate how well surface complexation models (SCMs) represent cell envelope reactivity. It also gives us the ability to determine site-specific enthalpies and entropies of protonation, which can aid both in site structure identification and description of conformational changes to those sites, further improving our understanding of the CN32 cell envelope. We gathered potentiometric and isothermal calorimetric titration data of CN32 over a range of ionic strength (0.02 – 0.53 M) while monitoring cell viability and performing Fourier transform infrared spectroscopy (FTIR) investigations. FTIR analysis was performed over a range of pH (4 – 9.4) to confirm identification of proton-active sites. Surface complexation modeling revealed that models involving 3 and 4 sites could adequately describe the potentiometric titration data; however, a 3-site model best represented the calorimetric data. We inferred the following site identities based on characteristic enthalpies, entropies, and equilibrium constants of protonation: carboxyl (log(K): 4.79 – 4.95), phosphoryl (6.69 – 6.97), and amine groups (9.37 – 9.64). Spectral corroboration via FTIR indicated the presence of chemical moieties consistent with these functional group assignments. Ionic strength was shown to have a significant impact on the concentration of all three sites at 95% confidence. Entropies and equilibrium constants of protonation for carboxyl and phosphoryl sites did not significantly change with ionic strength. For the carboxyl group, enthalpies of protonation varied significantly between the lowest and highest ionic strengths examined, though enthalpies at intermediate concentrations were not distinguishable. Enthalpies of protonation for the amine group were statistically different at each ionic strength. Additionally, entropies and equilibrium constants of protonation for the amine group were significantly affected by all ionic strengths tested. The entropy of protonation for the amine site changed from −21.5 ± 6.1 to +109.0 ± 4.2 J/molK−1 as ionic strength increased from 0.02 to 0.53 M, respectively. A change in the entropy of protonation of this magnitude and sign is indicative of a substantial conformational change in this functional group. Cell viability was significantly impacted over the course the titrations; however, viability did not impact SCMs results. In sum, these data fill gaps in our understanding of CN32 and provide new insights into the intricacies of its cell envelope.