Ultrafast Structural Changes Decomposed from Serial Crystallographic Data
Authors: Ren Zhong 
Published: May 2019

Direct visualization of electronic and molecular events during chemical and biochemical reactions will offer unprecedented mechanistic insights. Ultrashort pulses produced by X-ray free electron lasers (XFELs) offer an unprecedented opportunity for direct observations of transient events as short-lived as tens of femtoseconds. This paper presents an in-depth analysis of the serial crystallographic datasets collected by Barends & Schlichting et al. (Science 350, 445, 2015) that probe the ligand photodissociation in carbonmonoxy myoglobin (MbCO), a long-serving hallmark for observing ultrafast dynamics in a biological system. This analysis reveals electron density changes that are caused directly by the formation of high-spin 3datomic orbitals of the heme iron upon the CO departure and the dynamic behaviors of these newly formed orbitals in a time series within the first few picoseconds. The heme iron is found vibrating at a high frequency and developing a positional modulation that causes the iron to pop out of and recoil back into the heme plane in succession. These findings here provide long-awaited visual validations for previous works using ultrafast spectroscopy and molecular dynamics simulations. This analysis also extracts electron density variations largely in the solvent during the first period of a low frequency oscillation previously detected by coherence spectroscopy. This work demonstrates the power and importance of the analytical methods in detecting and isolating transient, often very weak signals of electronic changes arising from chemical reactions in proteins.

Direct imaging of ultrafast and subtle structural events during a biochemical reaction, such as a single electronic transition from one atomic or molecular orbital to another, is highly desirable but has been beyond the reach of protein crystallography. It entails the capability of observing changes in electronic distributions at both an ultrafast time scale and an ultrahigh spatial resolution (Itatani et al., Nature 432, 867, 2004). The recent developments in femtosecond serial crystallography at X-ray free electron lasers (XFELs) have brought the achievable temporal resolution within a striking distance. This paper presents the electron density map decomposed from the XFEL data that shows the remanence of several 3datomic orbitals of the heme iron at an available spatial resolution although the map component is not an accurate image of the atomic orbitals. A key strategy that has enabled the findings here is a numerical deconvolution to resolve concurrent variations in a series of time-resolved electron density maps so that the electron densities influenced by an electron transfer event can be isolated as a partial change from the overwhelming presence of the bulk electrons that are not directly involved in bonding. Even at the limited spatial resolution, the subtle changes in electron distribution due to a spin crossover can be decoupled from far greater changes due to atomic displacements. Direct observations of electronic orbitals could offer unprecedented mechanistic insights into a myriad of chemical and biochemical reactions such as electron transfer in redox reactions, and formation, rupture, and isomerization of chemical bonds.

Ligand photodissociation in carbonmonoxy myoglobin (MbCO) has been a benchmark for studying ultrafast protein dynamics in a biological system. A number of studies in time-resolved crystallography have progressively improved the time resolution (from Šrajer et al., Science 274, 1726, 1996 to Barends et al., Science 350, 445, 2015). This paper presents an in-depth analysis of the serial crystallographic datasets of MbCO that Barends & Schlichting et al. (2015) contributed to the Protein Data Bank. First, a component of electron density distributions clearly shows the characteristic shape of the high-spin 3dorbitals reappeared at the heme iron upon the photodissociation of the CO ligand despite the limited accuracy of the orbital image due to the available spatial resolution. Second, the dynamic behaviors of these newly regained 3dorbitals within picoseconds after the photolysis provide long-awaited structural validation for previous spectroscopic observations and computational simulations. Specifically, the newly formed densities are oscillating with the heme iron at a high frequency of a thousand wavenumbers and developing a positional modulation during the first few picoseconds (Champion, Science 310, 980, 2005). The iron pops out of the heme plane at a few picoseconds and recoils back and pops out again afterwards. The dominant oscillation at a low frequency of several tens wavenumbers previously detected by coherence spectroscopy can be clearly resolved from the time series of electron density maps. The associated changes in electron density during the first cycle of the oscillation are largely located in the solvent rather than on the protein or heme, which suggests that the low frequency oscillations in a number of heme proteins, including MbCO, likely originate from a photolysis triggered pressure wave propagating in the solvated protein. Finally, these findings of chemical signals are isolated from coexisting thermal artifacts also by the numerical deconvolution. It is modeled in this study that the ultrashort XFEL pulses cause a transient spike of the local temperature at the heme site of hundreds of K.