Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

Distant Fe(2+)-Ru(3+) electronic couplings have been extracted from intramolecular electrontransfer rates in Ru(histidine(x)) (where X = 33, 39, 62, and 72) derivatives of cytochrome c. The couplings increase according to 62 (0.0060) < 72 (0.057) < 33 (0.097) < 39 (0.11 per wave numbers); however, this order is out of line with the histidine to heme edge-edge distances [62 (14.8) > 39 (12.3) > 33 (11.1) > 72 (8.4 angstroms)]. The rates (and the couplings) correlate with the lengths of sigma-tunneling pathways comprised of covalent bonds, hydrogen bonds, and through-space jumps from the histidines to the heme group. Space jumps greatly decrease couplings: One from Pro(71) to Met(80) extends the sigma-tunneling length of the His(72) pathway by roughly 10 covalent-bond units.

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

The rates of Ru(His33)cytochrome c electron-transfer (ET) reactions have been measured over a driving-force range of 0.59 to 1.89 eV. The driving-force dependence of Fe2+ → Ru3+ ET in RuL2(im)(His33)cyt c [L = 2,2‘-bipyridine (bpy), 4,4‘,5,5‘-tetramethyl-2,2‘-bipyridine (4,4‘,5,5‘-(CH3)4-bpy), 4,4‘-dimethyl-2,2‘-bipyridine (4,4‘-(CH3)2-bpy), 4,4‘-bis(N-ethylcarbamoyl)-2,2‘-bipyridine (4,4‘-(CONH(C2H5))2-bpy), 1,10-phenanthroline (phen); im = imidazole] is well described by semiclassical ET theory with k max = 2.7 × 106 s-1 (HAB = 0.095 cm-1) and λ = 0.74 eV. As predicted by theory, the rate of an exergonic (−ΔG° = 1.3 eV) heme reduction reaction, *Ru2+(bpy)2(im)(His) → Fe3+, falls in the inverted region (k = 2.0 × 105 s-1). In contrast, the rates of three highly exergonic heme reductions, *Ru2+(phen)2(CN)(His) → Fe3+ (2.0 × 105 s-1; 1.40 eV), Ru+(4,4‘-(CONH(C2H5))2-bpy)2(im)(His) → Fe3+ (2.3 × 105 s-1; 1.44 eV), and Ru+(phen)2(CN)(His) → Fe3+ (4.5 × 105 s-1; 1.89 eV), are much higher than expected for reactions directly to ground-state products. Agreement with theory is greatly improved by assuming that an electronically excited ferroheme (Fe2+ → *Fe2+; ∼ 1.05 eV) is the initial product in each of these reactions.

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2(+)-modified (bpy = 2,2'-bipyridine; im = imidazole) cytochrome c and azurin. A driving-force study with the His33 derivatives of cytochrome c indicates that the reorganization energy (lambda) for Fe2+-->Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex, *Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+-->Ru3+ and Cu+-->Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 A-1) that is consistent with prediction of the pathway model.