Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase.

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen-deuterium exchange experiments to define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nanosecond fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nanosecond and represents the enthalpic barrier to the reaction of SLO.

Artificial Metalloproteins: At the Interface between Biology and Chemistry.

Artificial metalloproteins (ArMs) have recently gained significant interest due to their potential to address issues in a broad scope of applications, including biocatalysis, biotechnology, protein assembly, and model chemistry. ArMs are assembled by the incorporation of a non-native metallocofactor into a protein scaffold. This can be achieved by a number of methods that apply tools of chemical biology, computational de novo design, and synthetic chemistry. In this Perspective, we highlight select systems in the hope of demonstrating the breadth of ArM design strategies and applications and emphasize how these systems address problems that are otherwise difficult to do so with strictly biochemical or synthetic approaches.

Detecting and Characterizing the Kinetic Activation of Thermal Networks in Proteins: Thermal Transfer from a Distal, Solvent-Exposed Loop to the Active Site in Soybean Lipoxygenase.

The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317-334) that correlate well with trends in the corresponding enthalpic barriers for kcat. A model for this behavior posits that collisions between water and loop 317-334 initiate thermal activation at the protein surface that is then propagated 15-34 Å inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317-334 (Q322C) or on a control loop, 586-603 (S596C). Chemical modification of cysteines with the fluorophore 6-bromoacetyl-2-dimethylaminonaphthalene (Badan, BD) provides site-specific probes for the measurement of fluorescence relaxation lifetimes and Stokes shift decays as a function of temperature. Computational studies indicate that surface water structure is likely to be largely preserved in each sample. While both loops exhibit temperature-independent fluorescence relaxation lifetimes as do the Stokes shifts for S596C-BD, the activation enthalpy for the nanosecond solvent reorganization at Q322C-BD (Ea(ksolv) = 2.8(0.9) kcal/mol)) approximates the enthalpy of activation for catalytic C-H activation (Ea(kcat) = 2.3(0.4) kcal/mol). This study establishes and validates the methodology for measuring rates of rapid local motions at the protein/solvent interface of SLO. These new findings, when combined with previously published correlations between protein motions and the rate-limiting hydride transfer in a thermophilic alcohol dehydrogenase, provide experimental evidence for thermally induced "protein quakes" as the origin of enthalpic barriers in catalysis.

Dynamical inversion of the energy landscape promotes non-equilibrium self-assembly of binary mixtures.

When driven out of equilibrium, many diverse systems can form complex spatial and dynamical patterns, even in the absence of attractive interactions. Using kinetic Monte Carlo simulations, we investigate the phase behavior of a binary system of particles of dissimilar size confined between semiflexible planar surfaces, in which the nanoconfinement introduces a non-local coupling between particles, which we model as an activation energy barrier to diffusion that decreases with the local fraction of the larger particle. The system autonomously reaches a cyclical non-equilibrium state characterized by the formation and dissolution of metastable micelle-like clusters with the small particles in the core and the large ones in the surrounding corona. The power spectrum of the fluctuations in the aggregation number exhibits 1/f noise reminiscent of self-organized critical systems. We suggest that the dynamical metastability of the micellar structures arises from an inversion of the energy landscape, in which the relaxation dynamics of one of the species induces a metastable phase for the other species.