Nature's use of the protein-cofactor assembly provides molecules of extraordinary functional potency and diversity. With a combination of state of the art spectroscopy, bio-organic synthetic methods, protein design and synthesis, and macromolecular simulation methods we are now in a unique position to understand in detail how natural protein- cofactor assemblies function, to design synthetic versions to emulate them, and even to design combinations with radically new properties. The program project brings together a group of people who can make particular contributions to the project. Molecular dynamics simulations combined with continuum electrostatic calculations carried out by Sharp will be used to determine the contribution of the protein to the electric field and the effect of this field on the cofactor, on metal ligands, on particular residues within the protein and the surrounding water structure. These effects also will be evaluated by direct time and frequency dependent spectral probes of the dynamic spectral shifts of electron and vibrational inhomogeneities, including applications involving the detection of single molecules (Hochstrasser and Vanderkooi). Novel heme derivatives synthesized by Therien combined with proteins synthesized by the Dutton group allow for design of particular protein-cofactor assemblies with desired geometries and electric fields around the cofactor. Kinetics and stability of the polypeptide chain will be addressed by DeGrado and McCafferty, using computational and rapid kinetic techniques. Theoretical and experimental analysis of the protein-cofactor structure, stability, dynamics and the resultant functional properties will be used to guide the design and synthesis, while the facile ability to design and synthesize desired protein- cofactor assemblies will be used to refine our theoretical methods and interpretation of the experimental analyses. We propose that the interplay between these different approaches will dramatically advance our understanding and use of protein-cofactors assemblies.