Histidine Orientation Modulates the Structure and Dynamics of a <i>de Novo</i> Metalloenzyme Active Site

The ultrafast dynamics of a <i>de novo</i> metalloenzyme active site is monitored using two-dimensional infrared spectroscopy. The homotrimer of parallel, coiled coil α-helices contains a His₃-Cu­(I) metal site where CO is bound and serves as a vibrational probe of the hydrophobic interior of the self-assembled complex. The ultrafast spectral dynamics of Cu-CO reveals unprecedented ultrafast (2 ps) nonequilibrium structural rearrangements launched by vibrational excitation of CO. This initial rapid phase is followed by much slower ∼40 ps vibrational relaxation typical of metal-CO vibrations in natural proteins. To identify the hidden coupled coordinate, small molecule analogues and the full peptide were studied by QM and QM/MM calculations, respectively. The calculations show that variation of the histidines’ dihedral angles in coordinating Cu controls the coupling between the CO stretch and the Cu–C–O bending coordinates. Analysis of different optimized structures with significantly different electrostatic field magnitudes at the CO ligand site indicates that the origin of the stretch–bend coupling is not directly due to through-space electrostatics. Instead, the large, ∼3.6 D dipole moments of the histidine side chains effectively transduce the electrostatic environment to the local metal coordination orientation. The sensitivity of the first coordination sphere to the protein electrostatics and its role in altering the potential energy surface of the bound ligands suggests that long-range electrostatics can be leveraged to fine-tune function through enzyme design

Pulse Electron Paramagnetic Resonance Studies of the Interaction of Methanol with the S₂ State of the Mn₄O₅Ca Cluster of Photosystem II

The binding of the substrate analogue methanol to the catalytic Mn₄CaO₅ cluster of the water-oxidizing enzyme photosystem II is known to alter the electronic structure properties of the oxygen-evolving complex without retarding O₂-evolution under steady-state illumination conditions. We report the binding mode of ¹³C-labeled methanol determined using 9.4 GHz (X-band) hyperfine sublevel-correlation (HYSCORE) and 34 GHz (Q-band) electron spin–echo electron nuclear double resonance (ESE-ENDOR) spectroscopies. These results are compared to analogous experiments on a mixed-valence Mn­(III)­Mn­(IV) complex (2-OH-3,5-Cl₂-salpn)₂­Mn­(III)­Mn­(IV) (salpn = <i>N</i>,<i>N</i>′-bis­(3,5-dichlorosalicylidene)-1,3-diamino-2-hydroxypropane) in which methanol ligates to the Mn­(III) ion (Larson et al. (1992) J. Am. Chem. Soc., 114, 6263). In the mixed-valence Mn­(III,IV) complex, the hyperfine coupling to the ¹³C of the bound methanol (<i>A</i>_iso = 0.65 MHz, <i>T</i> = 1.25 MHz) is appreciably larger than that observed for ¹³C methanol associated with the Mn₄CaO₅ cluster poised in the S₂ state, where only a weak dipolar hyperfine interaction (<i>A</i>_iso = 0.05 MHz, <i>T</i> = 0.27 MHz) is observed. An evaluation of the ¹³C hyperfine interaction using the X-ray structure coordinates of the Mn₄CaO₅ cluster indicates that methanol does not bind as a terminal ligand to any of the manganese ions in the oxygen-evolving complex. We favor methanol binding in place of a water ligand to the Ca²⁺ in the Mn₄CaO₅ cluster or in place of one of the waters that form hydrogen bonds with the oxygen bridges of the cluster