Vitamin B12 (or cobalamin) supports just two enzymes in the human proteome, but its absence is incompatible with life. This high value dietary commodity is escorted by an elaborate system of chaperones to its two client enzymes: methionine synthase (MS) in the cytoplasm and methylmalonyl-CoA mutase (MCM) in the mitochondrion. Clues to the complexity and multicompartment geography of the B12 trafficking pathway had emerged from clinical genetics studies on patients with inborn errors of cobalamin metabolism, which led to their classification into nine complementation groups (cblA-G, cblJ and mut). Immodest in size, translocation of this portly and complex organometallic cofactor, complete with a tail that is absent in other tetrapyrroles, presents challenges, both chemical and steric. With the discovery of the disease-associated cbl genes, the PI’s group has led the effort to decipher function, illuminating structural, spectroscopic, and kinetic details of the elegant redox- linked coordination chemistry that is integral to the trafficking pathway. Exciting advances in the past cycle combined with a wealth of provocative leads, position us to address major gaps in our understanding of B12 trafficking, which are fundamentally important and clinically relevant to isolated homocystinuria (due to a functional deficiency of MS) or methylmalonic aciduria (due to a functional deficiency of MCM) or both (due to mutations in the early steps in the trafficking pathway). Clinical data on patient mutations in the cbl and mut (MCM) genes, will continue to be integrated into our studies as we address the following specific aims in the next cycle. (i) Elucidate the mechanism by which chaperones in the shared cytoplasmic pathway direct B12 traffic at a branchpoint. Specifically, we will test our hypothesis that alternative interprotein Co-S complexes between CblC, a chemically versatile b-ligand transferase/eliminase, and CblD, which we discovered has two B12 bindings sites, govern partitioning between the cytoplasmic versus mitochondrial branches. We will also test the intriguing hypothesis that B12 piggybacks on CblD to enter the mitochondrion. (ii) Elucidate the mechanism of B12 transfer in the cytoplasmic branch from CblD to MS following our discovery that vicinal thiols are important for B12 delivery. We hypothesize that B12 tethered via a Co-S bond to CblD is loaded onto MS, and that the complex is freed via displacement of the thiolate ligand to form holo-MS and an intramolecular disulfide on CblD. (iii) Elucidate the molecular traffic lights in the mitochondrial branch that permit 5´-deoxyadenosylcobalamin loading from ATR (adenosyltransferase) to MCM and cob(II)alamin off-loading from MCM to ATR (for repair) in the presence of the G-protein chaperone, CblA. We will also assess the potential for rhodibalamin to form a rhodium-carbon bond (catalyzed by ATR) but resist its cleavage (by MCM). A small gene cluster, including ATR and MCM is essential for Mycob...