High-Field EPR Spectroscopic Characterization of Mn(II) Bound to the Bacterial Solute-Binding Proteins MntC and PsaA

Abstract

During infection, the bacterial pathogens Staphylococcus aureus and Streptococcus pneumoniae employ ATP-binding cassette (ABC) transporters to acquire Mn(II), an essential nutrient, from the host environment. Staphylococcal MntABC and streptococcal PsaABC attract the attention of the biophysical and bacterial pathogenesis communities because of their established importance during infection. Previous biophysical examination of Mn(II)-MntC and Mn(II)-PsaA using continuous-wave (≈9 GHz) electron paramagnetic resonance (EPR) spectroscopy revealed broad, difficult-to-interpret spectra (Hadley et al. J. Am. Chem. Soc. 2018, 140, 110-113). Herein we employ high-frequency (>90 GHz), high-field (>3 T) EPR spectroscopy to investigate the Mn(II)binding sites of these proteins and determine the Spin Hamiltonian parameters. Our analyses demonstrate that the zero-field splitting (ZFS) is large for Mn(II)-MntC and Mn(II)-PsaA at +2.72 and +2.87 GHz, respectively. The measured 55 Mn hyperfine coupling values for Mn(II)-MntC and Mn(II)-PsaA of 241 and 236 MHz, respectively, demonstrate a more covalent interaction between Mn(II) and the protein compared to Mn(II) in aqueous solution (≈265 MHz). These studies indicate that MntC and PsaA bind Mn(II) in a similar coordination geometry. Comparison of the ZFS values determined herein with those ascertained for other Mn(II) proteins suggests that the Mn(II)-MntC and Mn(II)-PsaA coordination spheres are not five coordinate in solution.

Crystal structure of imidazo[1,5-a]pyridinium-based hybrid salt (C13H12N3)2[MnCl4]

“…The EPR spectra of another hybrid, [(C 2 H 5 ) 4 N] 2 MnCl 4 , also consist of a broadened line with the isotropic g-value of 2.001 (3) in the temperature range 170-300 K (Ostrowski & Ciżman, 2008). The appearance of fine structure in the spectrum of (I) needs further study that requires the high-field/high-frequency EPR spectroscopy experiments to be undertaken at lower temperatures (Gagnon et al, 2019).…”

Comparing Nonbonded Metal Ion Models in the Divalent Cation Binding Protein PsaA

“…A recent high field EPR study stated that the Mn 2+ bound PsaA crystal structure could be consistent with bidentate carboxylate coordination, as the crystallographic metal carboxylate oxygen distances range from 2.1 to 2.4 Å. 57 Additionally, transition to the octahedral Mn-bound open conformation likely proceeds through six coordinate intermediates given the six coordinate geometry of the metal-bound open state (Figure 1F). Given the above, we suggest that octahedral coordination of Mn 2+ with bidentate coordination of Glu205 and Asp280 observed in simulations of Mn 2+ bound to PsaA may not be an artifact of the parameter sets used in this work.…”

“…Bidentate carboxylate coordination was the dominant water free coordination geometry of metal bound PsaA across all three studied ion models, in some contrast to the tetrahedral Mn 2+ crystal structure and in significant contrast to the tetrahedral Zn 2+ bound crystal structure. A recent high field EPR study stated that the Mn 2+ bound PsaA crystal structure could be consistent with bidentate carboxylate coordination, as the crystallographic metal carboxylate oxygen distances range from 2.1 to 2.4 Å . Additionally, transition to the octahedral Mn-bound open conformation likely proceeds through six coordinate intermediates given the six coordinate geometry of the metal-bound open state (Figure F).…”