Phosphorus Kβ X-ray emission spectroscopy detects non-covalent interactions of phosphate biomolecules in situ†

Phosphorus is ubiquitous in biochemistry, being found in the phosphate groups of nucleic acids and the energy-transferring system of adenine nucleotides (e.g. ATP). Kβ X-ray emission spectroscopy (XES) of phosphorus has been largely unexplored, with no previous applications to biomolecules. Here, the potential of P Kβ XES to study phosphate-containing biomolecules, including ATP and NADPH, is evaluated, as is the application of the technique to aqueous solution samples. P Kβ spectra offer a detailed picture of phosphate valence electronic structure, reporting on subtle non-covalent effects, such as hydrogen bonding and ionic interactions, that are key to enzymatic catalysis. Spectral features are interpreted using density functional theory (DFT) calculations, and potential applications to the study of biological energy conversion are highlighted.


The PINK X-ray Emission Spectrometer and Liquid Cell
The present experiments were performed at the PINK end station at the BESSY II synchrotron in Berlin. The end station was configured with a sample environment chamber (SEC) and a vacuum von Hamos spectrometer, separated by an 8-μm Kapton film window. The SEC contains a motorized sample stage to which multiple sample cells of various types can be mounted. Gas-tight liquid passthrough into and out of the SEC was achieved with standard HPLC fittings.
Due to the significant attenuation of 2 keV photons by air, the SEC and spectrometer were kept under vacuum at 5 mbar and 10 -5 mbar, respectively. With minor modifications, the SEC could be filled with one atmosphere of helium instead of vacuum, allowing for study of vacuumincompatible samples at a cost of ~2% of detection efficiency.
An enlarged diagram of the liquid cell is presented in Figure S1. The flow in/out ports, shown as black circles in the back plate, are recessed in an oval flow channel routed 1 mm deep into the back plate. Two quarter inch ports machined into the reverse side of the back plate allow connection of the liquid cell to standard HPLC components. The liquid cell is held together with four bolts. Figure S1. Enlarged view of the liquid sample cell.

Line Broadening in XES
P Kβ X-ray emission spectra are a sum of peaks, each with a Voigt profile. A Voigt function is a convolution of a Lorentzian and a Gaussian function, with respective broadening parameters of Γ and σ. The Gaussian broadening is due to the normal distribution of the spectrometer response function, while the Lorentzian broadening is due to the natural linewidth of the X-ray emission process. In a nonresonant X-ray emission experiment, the observed natural linewidth is the sum of the natural linewidths of the core-hole intermediate state and the valence-hole final state 1,2 : Γ XES = Γ P(1s) + Γ valence Campbell & Papp recommend a value of Γ P(1s) = 0.47 eV, corresponding to a lifetime of τ = 1.4 × 10 -15 s. 3 This value provides a lower bound for the Lorentzian broadening in non-resonant P K-edge XES in the limit of a long-lived final state.
The sharpest spectrum collected in the present study at the PINK beamline was that of NaPF 6 ( Figure S2). To investigate the broadening parameters, the Kβ 1,3 spectrum was fit to a sum of 4 or 5 Voigt functions constrained to have Γ ≥ 0.47 eV ( Figure S3). For the main peak, broadenings of Γ = 0.47 eV and σ = 0.26-0.27 eV (Gaussian FWHM = 0.61-0.64 eV) were consistently obtained. These values may be taken as upper bounds; thus σ spectrometer ≤ 0.26 eV and Γ XES = 0.47 eV.
Calculated spectra in this study are presented with broadenings of Γ = 0.47 eV and σ = 0.66 eV (Gaussian FWHM = 1.55 eV), with the increased value of σ chosen to obtain a better match with experimental phosphate spectra and to avoid visual clutter. The fact that a larger broadening is required to obtain good agreement between theory and experiment suggests that effects not accounted for in the DFT calculations (such as multi-electron transitions, which can be significant in the Kβ spectra of other elements 4-7 ) contribute to P Kβ emission. Figure S2. Kβ X-ray emission spectrum of NaPF 6 Figure S3. Voigt fits of the NaPF 6 Kβ 1,3 spectrum, with the broadening parameters of the most intense peak inset

Spectra of Na 2 HPO 4
Powder and solution P Kβ spectra of Na 2 HPO 4 are presented in Figure S4. The periodicallyoscillating background in the spectrum was a result of technical difficulties with the solution cell during data collection and does not reflect a general limitation of the apparatus. Figure S4. Powder and solution P Kβ spectra of Na 2 HPO 4 , with difference (solution -powder) 4. ATP and Ethyl-triphosphate calculated spectra P Kβ spectra are specifically sensitive to the local phosphate electronic structure, not that of whole molecule. To demonstrate the insensitivity of P XES to distal effects, equivalent computed spectra of ATP and ethyl triphosphate, in which the ribonucleotide is replaced with -CH 2 CH 3 , are presented in Figure S5. Figure S5. Calculated P Kβ spectra of ATP and ethyl triphosphate

NADP and NADPH Structures
Continuum-solvated DFT geometry optimizations of NADP and NADPH consistently converged to structures with a hydrogen bond from the nicotinamide to the phosphate with which it shares a ribose ( Figure S8). Figure S8. DFT-optimized structures of NADP (left) and NADPH (right), with intramolecular hydrogen bonds from nicotinamide circled