De novo design of multi-domain metalloenzymes

The course of evolution required the recombination of protein domains to perform ever-growing complex functions. The presence of an additional domain in a multi-domain protein expands, alters, or modulates the functionality with respect to the isolated one-domain protein.[1] Taking inspiration from Nature, artificial proteins have been engineered combining different domains to develop bioinspired molecular machines, able to respond to external stimuli.[2] Here, we report a new computational strategy to design de novo multi-domain proteins. The new methodology led to the first example of an artificial metalloenzyme, in which allostery was designed completely from scratch.[3,4] In particular, DF (Due Ferri), a diiron phenol oxidase domain, and PS (Porphyrin-binding Sequence), a zinc porphyrin binding domain, were selected as individual proteins to be combined and give DFP (Due Ferri Porphyrin).[5] The multiple junctions were identified to colocalize the two domains, and obtain a more extensive structural coupling between them. Noteworthy, DFP not only preserves the structural and functional properties of the parental proteins, but also shows a modulation in cooperation between the two domains. The catalytic characterization of 4-aminophenol oxidation demonstrated Michaelis-Menten kinetic in the phenoloxidase activity, and high-lightened a 4-fold tighter Km and a 7-fold decrease in kcat upon binding of the designed zinc porphyrin ZnP (Zn-meso-(trifluoromethyl)porphin). Molecular Dynamics simulations suggested that the presence of ZnP restrains the conformational freedom of a second-shell Tyr, that have been previously shown to largely affect the reactivity of the diiron center. Subsequently, the binding fitness of the zinc porphyrin was changed to investigate the bidirectionality of the allosteric regulation. In the presence of the different zinc porphyrin ZnDP (ZnDeuteroporphyrin IX), the ferroxidase and phenol oxidase activities were still. DFP3 showed an excellent affinity for ZnDP, only one order lower in magnitude compared to the designed ZnP. Most importantly, the ZnDP affinity was modulated by the presence of zinc bound to DFP3, showing a 3fold decrease in KD, and demonstrating the presence of a back-regulation. The photosensitizing properties of zinc porphyrin-DFP3 complexes were tested in the oxidation of the biological redox cofactor NADH. The photocatalytic characterization highlighted the paramount role of the protein scaffold not only in increasing the reaction rate, but also in protecting the zinc porphyrins from highly reactive species. The lower binding fitness of DFP3 towards ZnDP hindered this protection, enabling a major permeability of these species and leading to the zinc porphyrin photobleaching. The high reactivity and versatility of such systems are a promising starting point for the de novo design of artificial photosystems for the storage of light energy in chemical fuels. [6]