The ubiD/ubiX or the homologous fdc/pad genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone biosynthesis1–3 or microbial biodegradation of aromatic compounds4–6 respectively. Despite biochemical studies on individual gene products, the composition and co-factor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear7–9. We show Fdc is solely responsible for (de)carboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesised by the associated UbiX/Pad10. Atomic resolution crystal structures reveal two distinct isomers of the oxidized cofactor can be observed: an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with drastically altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. While 1,3-dipolar cycloaddition is commonly used in organic chemistry11–12, we propose this presents the first example of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

Conformational control limits most electron transfer (ET) reactions in biology, but we lack general insight into the extent of conformational space explored, and specifically the properties of the associated energy landscape. Here we unite electron-electron double resonance (ELDOR) studies of the diradical (disemiquinoid) form of human cytochrome P450 reductase (CPR), a nicotinamide adenine phosphate dinucleotide (NADPH)-linked diflavin oxidoreductase required for P450 enzyme reduction, with functional studies of internal ET to gain new insight into the extent and properties of the energy landscape for conformationally controlled ET. We have identified multiple conformations of disemiquinoid CPR, which point to a rugged energy landscape for conformational sampling consistent with functional analysis of ET using high-pressure stopped-flow, solvent, and temperature perturbation studies. Crystal structures of CPR have identified discrete "closed" and "open" states, but we emphasize the importance of a continuum of conformational states across the energy landscape. Within the landscape more closed states that favor internal ET are formed by nucleotide binding. Open states that enable P450 enzymes to gain access to electrons located in the FMN-domain are favored in the absence of bound coenzyme. The extent and nature of energy landscapes are therefore accessible through the integration of ELDOR spectroscopy with functional studies. We suggest this is a general approach that can be used to gain new insight into energy landscapes for biological ET mediated by conformational sampling mechanisms.

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Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes.

The conditions necessary for the formation of a monolayer and a bilayer of a mutated form (P499C) of human cytochrome P450 reductase on a Au(110)/electrolyte interface have been determined using a quartz crystal microbalance with dissipation, atomic force microscopy, and reflection anisotropy spectroscopy (RAS). The molecules adsorb through a Au-S linkage and, for the monolayer, adopt an ordered structure on the Au(110) substrate in which the optical axes of the dipoles contributing to the RAS signal are aligned roughly along the optical axes of the Au(110) substrate. Differences between the absorption spectrum of the molecules in a solution and the RAS profile of the adsorbed monolayer are attributed to surface order in the orientation of dipoles that contribute in the low energy region of the spectrum, a roughly vertical orientation on the surface of the long axes of the isoalloxazine rings and the lack of any preferred orientation in the molecular structure of the dipoles in the aromatic amino acids. Our studies establish an important proof of principle for immobilizing large biological macromolecules to gold surfaces. This opens up detailed studies of the dynamics of biological macromolecules by RAS, which have general applications in studies of biological redox chemistry that are coupled to protein dynamics.