The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation.

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Catalytic M Center of Copper Monooxygenases Probed by Rational Design. Effects of Selenomethionine and Histidine Substitution on Structure and Reactivity.

The M centers of the mononuclear monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-monooxygenase bind and activate dioxygen en route to substrate hydroxylation. Recently, we reported the rational design of a protein-based model in which the CusF metallochaperone was repurposed via a His to Met mutation to act as a structural and spectroscopic biomimic. The PHM M site exhibits a number of unusual attributes, including a His2Met ligand set, a fluxional Cu(I)-S(Met) bond, tight binding of exogenous ligands CO and N3-, and complete coupling of oxygen reduction to substrate hydroxylation even at extremely low turnover rates. In particular, mutation of the Met ligand to His completely eliminates the catalytic activity despite the propensity of CuI-His3 centers to bind and activate dioxygen in other metalloenzyme systems. Here, we further develop the CusF-based model to explore methionine variants in which Met is replaced by selenomethionine (SeM) and histidine. We examine the effects on coordinate structure and exogenous ligand binding via X-ray absorption spectroscopy and electron paramagnetic resonance and probe the consequences of mutations on redox chemistry via studies of the reduction by ascorbate and oxidation via molecular oxygen. The M-site model is three-coordinate in the Cu(I) state and binds CO to form a four-coordinate carbonyl. In the oxidized forms, the coordination changes to tetragonal five-coordinate with a long axial Met ligand that like the enzymes is undetectable at either the Cu or Se K edges. The EXAFS data at the Se K edge of the SeM variant provide unique information about the nature of the Cu-methionine bond that is likewise weak and fluxional. Kinetic studies document the sluggish reactivity of the Cu(I) complexes with molecular oxygen and rapid rates of reduction of the Cu(II) complexes by ascorbate, indicating a remarkable stability of the Cu(I) state in all three derivatives. The results show little difference between the Met ligand and its SeM and His congeners and suggest that the Met contributes to catalysis in ways that are more complex than simple perturbation of the redox chemistry. Overall, the results stimulate a critical re-examination of the canonical reaction mechanisms of the mononuclear copper monooxygenases.

Rational Design of a Histidine-Methionine Site Modeling the M-Center of Copper Monooxygenases in a Small Metallochaperone Scaffold.

Mononuclear copper monooxygenases peptidylglycine monooxygenase (PHM) and dopamine ß-monooxygenase (DBM) catalyze the hydroxylation of high energy C-H bonds utilizing a pair of chemically distinct copper sites (CuH and CuM) separated by 11 Å. In earlier work, we constructed single-site PHM variants that were designed to allow the study of the M- and H-centers independently in order to place their reactivity sequentially along the catalytic pathway. More recent crystallographic studies suggest that these single-site variants may not be truly representative of the individual active sites. In this work, we describe an alternative approach that uses a rational design to construct an artificial PHM model in a small metallochaperone scaffold. Using site-directed mutagenesis, we constructed variants that provide a His2Met copper-binding ligand set that mimics the M-center of PHM. The results show that the model accurately reproduces the chemical and spectroscopic properties of the M-center, including details of the methionine coordination, and the properties of Cu(I) and Cu(II) states in the presence of endogenous ligands such as CO and azide. The rate of reduction of the Cu(II) form of the model by the chromophoric reductant N,N'-dimethyl phenylenediamine (DMPD) has been compared with that of the PHM M-center, and the reaction chemistry of the Cu(I) forms with molecular oxygen has also been explored, revealing an unusually low reactivity toward molecular oxygen. This latter finding emphasizes the importance of substrate triggering of oxygen reactivity and implies that the His2Met ligand set, while necessary, is insufficient on its own to activate oxygen in these enzyme systems.

Incorporation of Ni2+, Co2+, and Selenocysteine into the Auxiliary Fe-S Cluster of the Radical SAM Enzyme HydG.

The radical SAM enzyme HydG generates CO- and CN--containing Fe complexes that are involved in the bioassembly of the [FeFe] hydrogenase active cofactor, the H-cluster. HydG contains a unique 5Fe-4S cluster in which the fifth "dangler" Fe and the coordinating cysteine molecule have both been shown to be essential for its function. Here, we demonstrate that this dangler Fe can be replaced with Ni2+ or Co2+ and that the cysteine can be replaced with selenocysteine. The resulting HydG variants were characterized by electron paramagnetic resonance and X-ray absorption spectroscopy, as well as subjected to a Tyr cleavage assay. Both Ni2+ and Co2+ are shown to be exchange-coupled to the 4Fe-4S cluster, and selenocysteine substitution does not alter the electronic structure significantly. XAS data provide details of the coordination environments near the Ni, Co, and Se atoms and support a close interaction of the dangler metal with the FeS cluster via an asymmetric SeCys bridge. Finally, while we were unable to observe the formation of novel organometallic species for the Ni2+ and Co2+ variants, the selenocysteine variant retains the activity of wild type HydG in forming [Fe(CO)x(CN)y] species. Our results provide more insights into the unique auxiliary cluster in HydG and expand the scope of artificially generated Fe-S clusters with heteroatoms.