The Liquid Jet Endstation for Hard X-ray Scattering and Spectroscopy at the Linac Coherent Light Source.

The ability to study chemical dynamics on ultrafast timescales has greatly advanced with the introduction of X-ray free electron lasers (XFELs) providing short pulses of intense X-rays tailored to probe atomic structure and electronic configuration. Fully exploiting the full potential of XFELs requires specialized experimental endstations along with the development of techniques and methods to successfully carry out experiments. The liquid jet endstation (LJE) at the Linac Coherent Light Source (LCLS) has been developed to study photochemistry and biochemistry in solution systems using a combination of X-ray solution scattering (XSS), X-ray absorption spectroscopy (XAS), and X-ray emission spectroscopy (XES). The pump-probe setup utilizes an optical laser to excite the sample, which is subsequently probed by a hard X-ray pulse to resolve structural and electronic dynamics at their intrinsic femtosecond timescales. The LJE ensures reliable sample delivery to the X-ray interaction point via various liquid jets, enabling rapid replenishment of thin samples with millimolar concentrations and low sample volumes at the 120 Hz repetition rate of the LCLS beam. This paper provides a detailed description of the LJE design and of the techniques it enables, with an emphasis on the diagnostics required for real-time monitoring of the liquid jet and on the spatiotemporal overlap methods used to optimize the signal. Additionally, various scientific examples are discussed, highlighting the versatility of the LJE.

YfeX - A New Platform for Carbene Transferase Development with High Intrinsic Reactivity.

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA).

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.