Fabrication of bio-electrode systems decorated with redox active biomolecules, flavins, is demonstrated. Exploiting the photochemistry and electrochemistry of flavins, we explored the photo-electrochemical activity of flavin-functionalized electrode systems to assess their potential utility for sustainable energy production. As model systems, lumiflavin and flavin adenine dinucleotide were immobilized on carbon electrodes by dropcasting and covalent grafting techniques. Activity of these bio-electrodes towards generation of O2 from H2O in 0.5 M potassium phosphate buffer at pH 7.1 was demonstrated. Irradiation of the electrode system with visible light led to increased activity of the electrodes with a 3-fold enhancement of oxidation of H2O.

We demonstrate a 15-fold enhancement of solid-state NMR signals via dynamic nuclear polarization (DNP) based on a stable, naturally occurring radical in a protein: the flavin mononucleotide (FMN) semiquinone of flavodoxin. The line width of flavodoxin’s EPR signal suggests that the dominant DNP mechanism is the solid effect, consistent with the field-dependent DNP enhancement profile. The magnitude of the enhancement as well as the bulk-polarization build-up time constant (τB) with which it develops are dependent on the isotopic composition of the protein. Deuteration of the protein to 85% increased the nuclear longitudinal relaxation time T1n and τB by factors of five and seven, respectively. Slowed dissipation of polarization can explain the 2-fold higher maximal enhancement than that obtained in proteated protein, based on the endogenous semiquinone. In contrast, the long τB of TOTAPOL-based DNP in nonglassy samples was not accompanied by a similarly important long T1n, and in this case the enhancement was greatly reduced. The low concentrations of radicals occurring naturally in biological systems limit the magnitude of DNP enhancement that is attainable by this means. However, our enhancement factors of up to 15 can nonetheless make an important difference to the feasibility of applying solid-state NMR to biochemical systems. We speculate that DNP based on endogenous radicals may facilitate MAS NMR characterization of biochemical complexes and even organelles, and could also serve as a source of additional structural and physiological information.

Flavins mediate a wide variety of chemical reactions in biology. To learn how one cofactor can be made to execute different reactions in different enzymes, we are developing solid-state NMR (SSNMR) to probe the flavin electronic structure, via the 15N chemical shift tensor principal values (δii). We find that SSNMR has superior responsiveness to H-bonds, compared to solution NMR. H-bonding to a model of the flavodoxin active site produced an increase of 10 ppm in the δ11 of N5, although none of the H-bonds directly engage N5, and solution NMR detected only a 4 ppm increase in the isotropic chemical shift (δiso). Moreover SSNMR responded differently to different H-bonding environments, as H-bonding with water caused δ11 to decrease by 6 ppm, whereas δiso increased by less than 1 ppm. Our density functional theoretical (DFT) calculations reproduce the observations, validating the use of computed electronic structures to understand how H-bonds modulate the flavin’s reactivity.

We have exploited 15N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His′, paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His′ that ligate FeII. These confirm that neither the Q69H nor the Q69E mutation strongly affects the FeII electronic structure, despite the 250 mV and > 660 mV increases in Em they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to FeII, as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to FeIISOD outside the FeII coordination sphere, affecting Gln69 but without direct involvement of His30.

Metalloenzymes catalyze some of the most demanding reactions in biochemistry, thereby enabling organisms to extract energy from redox reactions and utilize inorganic starting materials such as N2 and CH4. Bound metal ions bring to enzymes greater chemical versatility and reactivity than would be possible from amino acids alone. However the host proteins must control this broad reactivity, activating the metal for the intended reaction while excluding the rest of its chemical repertoire. To this end, metalloproteins must control the metal ion reduction midpoint potential (Em), because the Em determines what redox reactions are possible. We have documented potent redox tuning in Fe- and Mn-containing superoxide dismutases (FeSODs and MnSODs), and manipulated it to generate FeSOD variants with Ems spanning 900 mV (21 kcal/mol or 87 kJ/mol) with retention of overall structure. This achievement demonstrates possibilities and strategies with great promise for efforts to design or modify catalytic metal sites. FeSODs and MnSODs oxidize and reduce superoxide in alternating reactions that are coupled to proton transfer, wherein the metal site is believed to cycle between M3+·OH− and M2+·OH2 (M = Fe or Mn). Thus the Em reflects the ease both of reducing the metal ion and of protonating the coordinated solvent molecule. Moreover similar Ems are achieved by Fe-specific and Mn-specific SODs despite the very different intrinsic Ems of high-spin Fe3+/2+ and Mn3+/2+. We provide evidence that Em depression by some 300 mV can be achieved via a key enforced H-bond that appears able to disfavor proton acquisition by coordinated solvent. Based on 15N-nuclear magnetic resonance (NMR), stronger H-bond donation to coordinated solvent can explain the greater redox depression achieved by the Mn-specific SOD protein compared with the Fe-specific protein. Furthermore, by manipulating the strength and polarity of this one H-bond, with comparatively minor perturbation to active site atomic and electronic structure, we succeeded in raising the Em of FeSOD by more than 660 mV, apparently by a combination of promoting protonation of coordinated solvent and providing an energetically favorable source of a redox-coupled proton. These studies have combined the use of electron paramagnetic resonance (EPR), NMR, magnetic circular dichroism (MCD), and optical spectrophotometry to characterize the electronic structures of the various metal sites, with complementary density functional theoretical (DFT) calculations, NMR spectroscopy, and X-ray crystallography to define the protein structures and protonation states. Overall, we have generated structurally homologous Fe sites that span some 900 mV, and have demonstrated the enormous redox tuning accessible via the energies associated with proton transfer coupled to electron transfer. In this regard, we note the possible significance of coordinated solvent molecules in numerous biological redox-active metal sites besides that of SOD.

Superoxide dismutases (SODs) catalyze the de toxification of superoxide. SODs therefore acquired great importance as O2 became prevalent following the evolution of oxygenic photosynthesis. Thus the three forms of SOD provide intriguing insights into the evolution of the organisms and organelles that carry them today. Although ancient organisms employed Fe-dependent SODs, oxidation of the environment made Fe less bio-available, and more dangerous. Indeed, modern lineages make greater use of homologous Mn-dependent SODs. Our studies on the Fe-substituted MnSOD of Escherichia coli, as well as redox tuning in the FeSOD of E. coli shed light on how evolution accommodated differences between Fe and Mn that would affect SOD performance, in SOD proteins whose activity is specific to one or other metal ion.

Fabrication of bio-electrode systems decorated with redox active biomolecules, flavins, is demonstrated. Exploiting the photochemistry and electrochemistry of flavins, we explored the photo-electrochemical activity of flavin-functionalized electrode systems to assess their potential utility for sustainable energy production. As model systems, lumiflavin and flavin adenine dinucleotide were immobilized on carbon electrodes by dropcasting and covalent grafting techniques. Activity of these bio-electrodes towards generation of O2 from H2O in 0.5 M potassium phosphate buffer at pH 7.1 was demonstrated. Irradiation of the electrode system with visible light led to increased activity of the electrodes with a 3-fold enhancement of oxidation of H2O.