Modified His−His dipeptides have been reacted with copper(I) salts to model active-site Cu ions bound by contiguous His residues in certain oxygen-activating copper proteins, as well as amyloid β-peptide. Chelation of copper(I) by these ligands affords linear, two-coordinate complexes as studied structurally by X-ray absorption spectroscopy. The complexes are robust toward oxidation, showing limited to no reactivity with O2, and they bind CO weakly. Reaction with a third ligand (N-methylimidazole) affords complexes with a markedly different structure (distorted T-shaped) and reactivity, binding CO and oxidizing rapidly upon exposure to dioxygen.
Due to the growing need to provide alternatives to fossil fuels as efficiently, economically, and sustainably as possible there has been growing interest in improved biofuel production systems. Biofuels produced from microalgae are a particularly attractive option since microalgae have production potentials that exceed the best terrestrial crops by 2 to 10-fold. In addition, autotrophically grown microalgae can capture CO2 from point sources reducing direct atmospheric greenhouse gas emissions. The enhanced biomass production potential of algae is attributed in part to the fact that every cell is photosynthetic. Regardless, overall biological energy capture, conversion, and storage in microalgae are inefficient with less than 8% conversion of solar into chemical energy achieved. In this review, we examine the thermodynamic and kinetic constraints associated with the autotrophic conversion of inorganic carbon into storage carbohydrate and oil, the dominant energy storage products in Chlorophytic microalgae. We discuss how thermodynamic restrictions including the loss of fixed carbon during acetyl CoA synthesis reduce the efficiency of carbon accumulation in lipids. In addition, kinetic limitations, such as the coupling of proton to electron transfer during plastoquinone reduction and oxidation and the slow rates of CO2 fixation by Rubisco reduce photosynthetic efficiency. In some cases, these kinetic limitations have been overcome by massive increases in the numbers of effective catalytic sites, e.g. the high Rubisco levels (mM) in chloroplasts. But in other cases, including the slow rate of plastoquinol oxidation, there has been no compensatory increase in the abundance of catalytically limiting protein complexes. Significantly, we show that the energetic requirements for producing oil and starch relative to the recoverable energy stored in these molecules are very similar on a per carbon basis. Presently, the overall rates of starch and lipid synthesis in microalgae are very poorly characterized. Increased understanding of the kinetic constraints of lipid and starch synthesis, accumulation and turnover would facilitate the design of improved biomass production systems.
Copper-transporting ATPases (Cu-ATPases) ATP7A and ATP7B play an essential role in human physiological function. Their primary function is to deliver copper to the secretory pathway and export excess copper from the cell for removal or further utilization. Cells employ Cu-ATPases in numerous physiological processes that include the biosynthesis of copper-dependent enzymes, lactation, and response to hypoxia. Biochemical studies of human Cu-ATPases and their orthologs have demonstrated that Cu-ATPases share many common structural and mechanistic characteristics with other members of the P-type ATPase family. Nevertheless, the Cu-ATPases have a unique coordinate environment for their ligands, copper and ATP, and additional domains that are required for sophisticated regulation of their intracellular localization and activity. Here, we review recent progress that has been made in understanding the structure of Cu-ATPases from the analysis of their individual domains and orthologs from microorganisms, and speculate about the implications of these findings for the function and regulation of human copper pumps.
Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.
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