Experimental and DFT Investigations Reveal the Influence of the Outer Coordination Sphere on the Vibrational Spectra of Nickel-Substituted Rubredoxin, a Model Hydrogenase Enzyme

Abstract

Nickel-substituted rubredoxin (NiRd) is a functional enzyme mimic of hydrogenase, highly active for electrocatalytic and solution-phase hydrogen generation. Spectroscopic methods can provide valuable insight into the catalytic mechanism, provided the appropriate technique is used. In this study, we have employed multi-wavelength resonance Raman spectroscopy coupled with DFT calculations on an extended active-site model of NiRd to probe the electronic and geometric structures of the resting state of this system. Excellent agreement between experiment and theory is observed, allowing normal mode assignments to be made on the basis of frequency and intensity analyses. Both metal-ligand and ligand-centered vibrational modes are enhanced in the resonance Raman spectra. The latter provide information about the hydrogen bonding network and structural distortions due to perturbations in the secondary coordination sphere. To reproduce the resonance enhancement patterns seen for high-frequency vibrational modes, the secondary coordination sphere must be included in the computational model. The structure and reduction potential of the NiIIIRd state have also been investigated both experimentally and computationally. This work begins to establish a foundation for computational resonance Raman spectroscopy to serve in a predictive fashion for investigating catalytic intermediates of NiRd.

Driving Protein Conformational Changes with Light: Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase

“…The resonance Raman setup was similar to that described previously. 52 Briefly, the beam was focused onto the sample at a 135˚ backscattering angle using an f/4 parabolic focusing mirror. The scattered light was collimated by an f/0.8 UV-fused silica aspheric lens (Edmund Optics, Barrington, NJ), focused onto the 100 µm slit of an f/4.6 single-grating spectrograph equipped with a 1200 gr/mm grating blazed at 500 nm (Princeton Instruments Isoplane 320, Trenton, NJ), and imaged onto a Peltier-cooled CCD (Princeton Instruments Pixis 100B, Trenton, NJ).…”

Light‐Driven Hydrogen Evolution by Nickel‐Substituted Rubredoxin

“…As is evident in Figure , both quenching pathways are thermodynamically accessible, with a strong driving force both for quenching of the excited *Ru state and subsequent back electron transfer, regenerating the ground state. In contrast, NiRd can access both the Ni II/I couple, which is thought to be the catalytically relevant transition at approximately −0.944 V vs. NHE at pH 6.5, along with a quasi‐reversible Ni III/II couple at 0.490 V, as measured by solution‐phase electrochemical experiments . Given the estimated potentials for the Ru phototrigger, only the reductive quenching pathway by Ni II would be thermodynamically accessible, with favorable back electron transfer to regenerate the resting state (Figure ).…”

“…At these concentrations, it is likely that, following photoexcitation, direct quenching of the *Ru excited state by ascorbate to generate Ru I is the dominant pathway (Figure S12). From this state, electron transfer to Ni II would be thermodynamically favorable, forming the Ni I state that could be protonated, reduced again, and ultimately evolve H 2 . To further investigate these processes, additional time‐resolved experiments, including transient absorption spectroscopy and pump‐probe EPR studies, will be required to resolve the timescales of electron transfer beyond the initial excitation event and identify intermediates.…”

Light‐driven catalysis with engineered enzymes and biomimetic systems

“…Although most hydrogenase mimicry has focused on the diiron hydrogenase, one of the earliest systems was based on the [NiFe] hydrogenase [155][156][157][158][159][160][161]. Substitution of Ni in rubredoxin yields a catalyst, Ni-Rd, with a tetrathiolate active site mimicking the primary coordination sphere of the Ni site in [NiFe] hydrogenase [155,161].…”