Hyperfine-shifted 13C resonance assignments in an iron-sulfur protein with quantum chemical verification: aliphatic C-H···S 3-center-4-electron interactions

Abstract

Although the majority of noncovalent interactions associated with hydrogen and heavy atoms in proteins and other biomolecules are classical hydrogen bonds between polar N-H or O-H moieties and O atoms or aromatic π electrons, high-resolution X-ray crystallographic models deposited in the Protein Data Bank show evidence for weaker C-H···O hydrogen bonds, including ones involving sp(3)-hybridized carbon atoms. Little evidence is available in proteins for the (even) weaker C-H···S interactions described in the crystallographic literature on small molecules. Here, we report experimental evidence and theoretical verification for the existence of nine aliphatic (sp(3)-hybridized) C-H···S 3-center-4-electron interactions in the protein Clostridium pasteurianum rubredoxin. Our evidence comes from the analysis of carbon-13 NMR chemical shifts assigned to atoms near the iron at the active site of this protein. We detected anomalous chemical shifts for these carbon-13 nuclei and explained their origin in terms of unpaired spin density from the iron atom being delocalized through interactions of the type: C-H···S-Fe, where S is the sulfur of one of the four cysteine side chains covalently bonded to the iron. These results suggest that polarized sulfur atoms in proteins can engage in multiple weak interactions with surrounding aliphatic groups. We analyze the strength and angular dependence of these interactions and conclude that they may contribute small, but significant, stabilization to the molecule.

Figure 1

Example of selective labeling used in assigning the ¹³C NMR signals from Clostridium pasteurianum rubredoxin with anomalous chemical shifts. (a) Reduced rubredoxin labeled with [U-13C]-valine. (b) Oxidized rubredoxin labeled with [U-13C]-valine. (c) Reduced rubredoxin labeled with [U-13C]-tyrosine. (d) Oxidized rubredoxin labeled with [U-13C]-tyrosine. The data collection method was optimized such that signals from nuclei close to the Fe−S center were observed preferentially in these spectra. The annotated ¹³C signals have unusual chemical shifts when compared to the diamagnetic averages obtained from BMRB.(19) We attribute these large shifts to participation of these carbons and their attached hydrogens in 3c−4e interactions with sulfur atoms coordinated to iron: CH···S−Fe.

Figure 2

Deviations of eight assigned ¹³C signals from reduced Clostridium pasteurianum rubredoxin from their mean diamagnetic chemical shifts. The error bars represent ±5 standard deviations from the mean diamagnetic shifts in BioMagResBank.(19) Residue and atom assignments are given along the abscissa. The total number of covalent bonds separating the iron atom and each carbon is shown in parentheses. The range of hyperfine shifts reported here corresponds to ¹³C hyperfine coupling constants of approximately 0.02−0.1 MHz.

Figure 3

Schematic representation of hydrogen bonds and 3c−4e interactions at the active site of Clostridium pasteurianum rubredoxin. The C−H···S 3c−4e interactions reported here (red arrows) were detected by 3c−4e Fermi-contact shifts on ¹³C. The N−H···S hydrogen bonds (blue arrows) were detected previously(31) by trans-hydrogen-bond Fermi-contact effects on ¹⁵N chemical shifts.

Figure 4

Robust linear regression fit of the DFT theoretical versus experimental chemical shifts (Table 1) for the eight anomalously shifted ¹³C NMR signals of rubredoxin identified in Figure 2. The outlier data point (○) had a small weight (5%) in the robust fit. All data represented by “●” had weights of greater than 90%. The squared regression coefficient of the fit, excluding the outlier, was R² = 0.98 (see text for more).

Figure 5

Plot of the dependence of the ¹³C hyperfine shift of CH₄ on the S···H distance and the Fe−S···H angle in the CH₄−Fe(II)[SCH₃]₄ complex. The angle and distance were varied systematically; these values were fixed, and all other degrees of freedom were optimized at each point. The level of theory for optimization was B3LYP/6-31+G(d). The hyperfine shifts were calculated from the partially optimized structures at the B3LYP/6-311G(d,p) level of theory.

Figure 6

Three-dimensional representation of the natural bond orbitals (NBOs) for hydrogen bonds to the sulfur atom of one of the four cysteine residues (cysteine-6) that coordinate the iron atom of rubredoxin (left). The antibonding iron d-orbital is in red and rust; the sulfur lone pair p-orbital is in yellow and orange. Shown are antibonding orbitals of one carbon−hydrogen 3c−4e interaction (green, valine-8 C^β−H) and two nitrogen−hydrogen bonds (blue, valine-6 N−H and cysteine-9 N−H) that are involved in bonding interactions with the sulfur lone pair. The figure is based on the spin densities extracted(18) from a Gaussian 03(17) calculation on the 209-atom model derived from the 1.5 Å crystal structure of reduced CpRd (PDB code 1FHM). The contour plot shows the overlap of orbitals involved in the 3e−4c interaction referred to in this publication (right). The contour plot was created with NBOView 1.0,(32) and the space-filling model was produced with gOpenMol^, from a Gaussian 03(17) cube file.