This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Methylamine dehydrogenase (MADH) is an ancient bacterial metabolic enzyme that evolved in a pre-aerobic world. Its anaerobic origins are attested to by the highly evolved multistep chemistry it catalyzes, and its use of ancillary redox protein partners, rather than molecular oxygen, to remove electrons during catalysis. The energy generated through these types of electron transfer reactions underlies all energy generation in living organisms. Although a product of convergent evolution, the reductive chemistry catalyzed by the enzyme is analogous to that of the copper-containing amine oxidase family, whose human counterparts are linked to late-diabetic complications and vascular changes in congestive heart disease. Using a novel combination of single crystal visible spectroscopy, X-ray crystallography and freeze-trapping, and small-angle X-ray scattering, this project will probe how the protein controls its complex multistep reaction at atomic resolution by trapping catalytic intermediates in Paracoccus denitrificans MADH (PD-MADH) containing crystals, and determining their structures. By studying this highly developed metabolic system, a part of the ancient anaerobic redox chemistries still found in mitochondria and chloroplasts today, we can shed light on the fundamental processes of harnessing energy and materials that evolved complex life on earth.