Abstract
X-ray absorption spectroscopy at the L-edge of 3d transition metals provides unique information on the local metal charge and spin states by directly probing 3d-derived molecular orbitals through 2p-3d transitions. However, this soft x-ray technique has been rarely used at synchrotron facilities for mechanistic studies of metalloenzymes due to the difficulties of x-ray-induced sample damage and strong background signals from light elements that can dominate the low metal signal. Here, we combine femtosecond soft x-ray pulses from a free-electron laser with a novel x-ray fluorescence-yield spectrometer to overcome these difficulties. We present L-edge absorption spectra of inorganic high-valent Mn complexes (Mn ∼ 6–15 mmol/l) with no visible effects of radiation damage. We also present the first L-edge absorption spectra of the oxygen evolving complex (Mn4CaO5) in Photosystem II (Mn < 1 mmol/l) at room temperature, measured under similar conditions. Our approach opens new ways to study metalloenzymes under functional conditions.
Highlights
We note that the concept we describe here is applicable to other 3d transition metals by adapting the optical properties of the reflective zone plates (RZPs) structures
We demonstrate the feasibility of Mn L-edge x-ray absorption (XAS) of dilute high-valent Mn complexes and Photosystem II (PS II) protein samples in solution with femtosecond soft x-ray pulses from an x-ray free-electron laser (XFEL)
Our results show that under our experimental conditions, the samples were probed without dose-dependent x-ray damage
Summary
Many important redox-active metalloenzymes such as Photosystem II (PS II), hydrogenases, and nitrogenases employ 3d transition metals in their active sites, where they catalyze multi-electron reactions in aqueous solution, at ambient temperature and pressure. While these catalysts cannot be transferred into industrial processes, they provide unique information on how to spatially and temporally control electron and proton flow and product/substrate transport during chemical transformations. Biological soft x-ray spectroscopy can take advantage of XFELs to collect x-ray damage-free data at room temperature by outrunning the sample damage with fs pulses if a suitable detection scheme is used Such a detection scheme needs to extract the La,b fluorescence signal (400–1000 eV) arising from the dilute metal sites and separate it from the very strong background from Ka fluorescence (277–525 eV), emitted by light elements in the sample (C, N, and O). Upon sequential absorption of visible photons, it advances in a series of intermediate states S0 ! S1 ! S2 ! S3 ! S4 and accumulates four oxidizing equivalents (unit charges) in the Mn4CaO5 cluster. We interpret Mn Ledge spectra of the Mn4CaO5 cluster in PS II in the dark resting state and in an illuminated state by comparing them to experimental spectra of structurally well-characterized Mn model complexes, measured under similar conditions
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