Influence of pump laser fluence on ultrafast structural changes in myoglobin

Abstract

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern whether this experimental approach allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments such that biologically relevant insight emerges.

From femtoseconds to minutes: time-resolved macromolecular crystallography at XFELs and synchrotrons

“…Compared with cryo-trapping, time-resolved crystallography appears to be the better-suited approach to probe a reaction since the conditions are closer to the physiological conditions. However, these experiments, which are most often performed in a serial manner, are challenging in terms of sample quantity, experimental knowhow (particularly the control of laser fluence) and diffraction data processing (Barends, Bhattacharyya et al, 2022). Also, diffraction resolution is often limited at room temperature.…”

The TR-icOS setup at the ESRF: time-resolved microsecond UV–Vis absorption spectroscopy on protein crystals

“…pump laser could affect the photoreaction and potentially lead to artefacts in the interpretation of structural intermediates (Miller et al, 2020;Barends et al, 2022). Power-titration spectroscopic experiments prior to the diffraction experiment should help to identify these potential artefacts.…”

Structural Dynamics of Metalloproteins and Redox Enzymology with Mix-and-Inject Time-Resolved Serial Femtosecond Crystallography

“…This knowledge could eventually facilitate the optimal design of artificial catalysts, for instance, by mimicking the energy transfer process in photosystems to ultimately yield comparable efficiencies, thereby facilitating the transformation of this biomimicking technology to industrial scales. , A reliable biochemical understanding of the function or catalysis of these metalloproteins, i.e., metalloenzymes, can be achieved by collecting tr -SFX diffraction images before the propagation of radiation damage in the vicinity of metal cofactors using the so-called “short-pulse duration” (few femtoseconds) and, more importantly, enabling SFX data collection at near-physiological temperatures, thus allowing reliable interpretations of their biological functions. The feasibility of using tr -SFX for studying metalloproteins has been successfully demonstrated using a pump–probe approach in several systems that are light sensitive, including photosystems, ,− , cytochrome c oxidases ( Cc O), , cytochrome P450 NO reductase, flavoenzymes such as DNA photolyases, and various heme proteins. , Nonetheless, only a few of these studies have implemented diffusion-based tr -SFX to elaborate on the dynamics of metalloproteins or redox enzymes. ,− …”