Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Abstract

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp 17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

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

“…Isomorphous difference maps of |F 15s obs -F free obs |, calculated using the structure factor phases of the enzyme-free state (φ free ), showed widely distributed conformational changes across ICH upon cysteine (Cys101) modification, i.e., thioimidate intermediate formation between the sulfhydryl group of Cys101 and the p-NPIC substrate, indicating the importance of these changes in ICH catalysis. 83,84 A drop-on-demand tape-drive-based sample delivery method was used to collect tr-SFX data on isopenicillin N-synthase (IPNS) with catalytic ferrous ions (Fe 2+ ), revealing the catalytic events of the reaction of L-δ-(α-aminoadipoyl)-Lcysteinyl-D-valine (ACV) with dioxygen to form isopenicillin (IPN), the precursor for all penicillin and cephalosporin antibiotics. 82 ACV substrate-bound fully reduced (IPNS:Fe-(II):ACV) anaerobic microcrystal slurries were exposed to atmospheric O 2, and redox intermediates were probed with Xrays at different delay times from 400 ms to 3.0 s via simultaneous collection of tr-SFX and tr-XES (time-resolved X-ray emission spectroscopy) data using a similar setup to that used for photosystem II.…”

“…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. ,− …”

“…Such information offers direct insight into redox enzyme catalysis when combined with individual redox state structures, i.e., fully reduced and oxidized states, as has been demonstrated for Cc O and the NADPH-cytochrome P450 reductase. ,,, By using MISC tr -SFX, it was also possible to observe the gating conformational dynamics of the redox hydrolase enzyme isocyanide hydratase (ICH) during catalysis, resolving a set of correlated conformational changes, i.e., nonequilibrium protein motions, that alter the ICH active site and increase catalytic efficiency . ICH catalyzes the irreversible reaction of isocyanide hydration to form N-formamide from a cysteine-derived thioimidate intermediate. , Microcrystals of ICH were mixed with a para-nitrophenyl isocyanide ( p -NPIC) substrate using a coMESH microfluidic T-junction mixing injector to the X-ray interaction region after 15 s and 5 min delay times. Isomorphous difference maps of |F 15s obs -F free obs |, calculated using the structure factor phases of the enzyme-free state (φ free ), showed widely distributed conformational changes across ICH upon cysteine (Cys101) modification, i.e., thioimidate intermediate formation between the sulfhydryl group of Cys101 and the p -NPIC substrate, indicating the importance of these changes in ICH catalysis. , …”

“…ICH catalyzes the irreversible reaction of isocyanide hydration to form N-formamide from a cysteine-derived thioimidate intermediate. , Microcrystals of ICH were mixed with a para-nitrophenyl isocyanide ( p -NPIC) substrate using a coMESH microfluidic T-junction mixing injector to the X-ray interaction region after 15 s and 5 min delay times. Isomorphous difference maps of |F 15s obs -F free obs |, calculated using the structure factor phases of the enzyme-free state (φ free ), showed widely distributed conformational changes across ICH upon cysteine (Cys101) modification, i.e., thioimidate intermediate formation between the sulfhydryl group of Cys101 and the p -NPIC substrate, indicating the importance of these changes in ICH catalysis. , …”