A biosynthetic approach to understanding the assembly of CuA centers
Electron transfer plays a critical role in countless biological processes. Metalloproteins containing copper or iron carry out electron transfer throughout all domains of life. CuA is a unique dinuclear, mixed-valence and valence delocalized copper center, which catalyzes remarkably efficient electron transfer under the low driving forces experienced at the termini of electron transport chains. Historically, CuA centers have been difficult to study, due to their occurrence in large enzyme complexes that are embedded in membranes and/or the presence of other metallocofactors in the same enzyme. For these reasons, soluble proteins containing only the CuA cofactor were created, either by truncating helices that anchored the CuA domain to a membrane, or by engineering the CuA center into another protein. The strategy of engineering CuA centers into another protein falls under the umbrella of the biosynthetic approach to metalloenzyme studies. In the biosynthetic approach to metalloenzyme studies, the protein is considered to be a large ligand to the metal center, which is customizable to the needs of that center. The biosynthetic approach offers many distinctive benefits in the study of native metal centers over the complementary approach of studying these centers in their native enzymes. These benefits include simpler protein systems, which are easy to express and purify in high yield and are free of other metal cofactors, and the ability to substitute non-native metals into the site. A biosynthetic model of CuA centers was previously designed into a mononuclear copper enzyme called azurin (Az). In this model, the CuA center was engineered into the same location of the protein as the original copper center, by replacement of a single loop containing a majority of the copper ligands. The resulting CuAAz construct binds copper in a copper site that is very similar to native CuA centers, as evidenced by many spectroscopic and structural studies. The ease of purification, amenability to mutagenesis, and stability in solution of CuAAz has resulted in many important insights into the properties of native CuA centers, such as a direct demonstration of the greater electron efficiency of CuA centers compared to mononuclear type 1 (T1) copper electron transfer sites. Some unresolved questions about native CuA enzymes are how these centers acquire copper ions and by what mechanism are these copper ions assembled into a functional CuA unit. Copper ions are tightly regulated in living systems, due to their capacity to generate hydroxyl radicals and their superior affinity for the ligand sets of other metals. Proteins, called copper iii chaperones, carry and deliver copper ions to their biological targets. One such copper chaperone, named Sco, has been proposed to deliver copper to the CuA center of cytochrome c oxidase. Sco binds both Cu(I) and Cu(II), supporting a copper chaperone function. However, such a function has yet to be confirmed for Sco proteins, and other redox regulatory functions have been offered as alternatives. Thus, the source of copper ions and the mechanism of their incorporation into CuA centers remains a mystery. Studies of in vitro Cu(II) incorporation into the biosynthetic CuA site of CuAAz have yielded some of the first reported mechanisms for CuA assembly. A previous study of Cu(II) incorporation into CuAAz under excess copper conditions showed the formation of a single T2 copper intermediate, which then converted directly to CuA. As reducing equivalents were required, but not supplied, to form the [Cu(1.5)Cu(1.5)] resting state of CuA, reduction of Cu(II) to Cu(I) by formation of a disulfide between the CuA Cys thiols was proposed. Increased CuA formation with the addition of exogenous reductant or Cu(I) supported this hypothesis. In this dissertation, a detailed mechanistic study of Cu(II) incorporation into CuAAz is described, in which the unmediated assembly of the CuA center is followed by pH- and oxygen-dependent standard and stopped-flow UV-Vis absorbance spectroscopy, as well as freeze-quench and standard EPR spectroscopy. Analysis of mutant CuAAz proteins, in which various of the copper-binding ligands were removed or perturbed, provided information about the ligand sets of the intermediates that formed during assembly of the CuA center. Under sub-equivalent copper concentrations, it is discovered that copper incorporation into the CuA center proceeds through two pathways. First, a type 2 (T2) copper capture complex forms, analogous to the sole intermediate observed in the previous study of CuA formation in CuAAz. In this study, it was discovered that this T2 copper capture complex formed between copper ions from bulk solution and Cys116 of the CuA site. This T2 copper intermediate then displayed two types of reactivity. In one, it reacted through a bimolecular pathway, involving the formation of Cu(I), to generate CuA, in an analogous pathway to that observed previously. In the other pathway, which had not been reported before, the T2 copper intermediate structurally rearranged to another intermediate, with unusual spectroscopic properties, called IX. Comparison to documented copper-thiolate complexes led us to propose that IX is a copper dithiolate complex, formed with both of the CuA Cys residues. IX then seemingly converted to a T1 copper complex. This T1 copper intermediate displayed ligation by Cys 112, as revealed through UV- iv Vis and EPR spectral characterizations. Spin quantification by EPR spectroscopy revealed that IX reacts to generate Cu(I) in the system. Oxygen-dependent stopped-flow UV-Vis absorbance spectroscopy showed that the formation of the T1 copper intermediate was dependent on oxygen, from which it was suggested that the T1 copper intermediate is a one-electron oxidation product of a Cu(I) species, generated by decay of IX. Finally, the T1 copper intermediate slowly converted to CuA, by reaction with Cu(I) in solution. The formation of these intermediates was also influenced by pH, with lower pH resulting in faster kinetics and less accumulation of the intermediates. This mechanism provides a picture of unmediated Cu(II) incorporation into a CuA center. The overall yield of this complex process was only ~30% of total expected yield, which was attributed to oxidation of the thiols of the CuA center to disulfide, coupled to the generation of Cu(I). Thus, while unmediated assembly of CuA centers from Cu(II) ions does result in fully-formed CuA sites, the efficiency of this metalation method is low. Nature, however, has evolved proteins for the sole purpose of maintaining a thiol-pair/disulfide in the correct oxidation state, called thioredoxins. Therefore, while metalation of CuA through Cu(I) or a mixture of Cu(I) and Cu(II) may be more efficient, metalation of CuA through Cu(II) and thioredoxin presents a practical scenario. Another biosynthetic take on the problem of CuA assembly is to try to understand how protein-mediated metalation of this center may occur. Toward this goal, a mutant of Az, H46C/C112H, which binds Cu(II) in a site very similar to a Sco variant, has been characterized. Importantly, while it has been demonstrated that the Cu(II) site of Sco is essential for its function in CcO maturation, no Cu(II)-bound Sco structure has been obtained. Despite only exchanging the positions of two copper-binding residues of the original T1 copper site, H46C/C112H Az forms a T2 copper center. The T2 copper center of this mutant exhibits pH- and time-dependent changes, which were investigated by UV-Vis and EPR spectroscopies. At pH 9, the UV-Vis and EPR spectra of this Az variant are nearly identical to those of a Sco mutant, C45A. Conditions where H46C/C112H Az forms crystals have been found, and a diffracted crystal structure is under refinement. Structural characterization of a Sco-like center in Az could provide the first such structure of a Sco-like Cu(II) center. Future designs of this Az mutant will incorporate all of the features of native Sco, which could both lead to a full structural description of these types of sites, as well as yield functional insight into Sco proteins.
biological electron transfer, CuA, CuA assembly, Sco, copper chaperones, metalloprotein design, azurin
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign