Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. The human metallo-chaperone protein Atox1 (known also as Hah1) delivers Cu I to the trans-Golgi network (1, 2). Atx1, the version from the yeast Saccharomyces cerevisiae, was the first copper metallo-chaperone to be identified (3). They both feature the classic ferredoxin ␤␣␤␤␣␤-fold with a CXXC motif acting as a high affinity Cu I -binding site ( Fig. 1) (4, 5). Homologues are found in cyanobacteria (Atx1), in Enterococcus hirae (CopZ), in Bacillus subtilis (CopZ), and in many other organisms (6).The human P 1B -type ATPase ATP7A accepts copper from Atox1 and transports it into the lumen of the trans-Golgi network (2). ATP7B performs a related role in liver cells. The inherited disorders Menkes and Wilson diseases are associated with defects in ATP7A and ATP7B, respectively (7). Equivalent metal transporters exist in other organisms such as Ccc2 from S. cerevisiae (3) and heavy metal ATPases 5-8 (HMA5-8) in the simple plant Arabidopsis thaliana (8). Their N termini contain between one and six metal-binding domains (MBDs) 2 that may interact with and receive Cu I directly from Atox1-type metallo-chaperones (6). It appears that, for some Cu I -ATPases at least, metal-binding sites in the transmembrane domain may also independently receive Cu I from copper chaperones (9). The overall molecular structure and binding site of each MBD is similar to that of Atox1 (10).Accurate estimation of affinities for Cu I (as expressed by the dissociation constant K D ) is essential for a quantitative understanding of reactivity and mechanisms of action. Yet reported K D values are scattered widely as highlighted by those of Atox1-type proteins, which differ by more than 10 orders of magnitude (K D ϳ10 Ϫ5 , 10 Ϫ10 , 10 Ϫ14 , and 10 Ϫ18 M) even though the structures and metal-binding sites of these proteins essentially superimpose (11-16). The various values were determined via different experimental approaches with different ligand probes and affinity standards. The affinities of some of the probes and standards remain in dispute. In addition, the intrinsic instability of free Cu ϩ in aqueous solution and the tendency to aerial oxidation of cysteine ligands impose special conditions on these experiments. These aspects are complicated further by reports that thiol ligands such as endogenous glutathione (GSH) may expand the Cu I coordination sphere in these proteins or lead to polymeric forms (14,17,18).In an attempt to resolve these fundamental issues for this iconic set of proteins, this study surveys the literature values for the Cu I affinities of the four probe ligands bicinchoninate (Bca), bathocuproine disulfonate (Bcs), dithiothreitol (Dtt), and glutathione (GSH) (Scheme 1). By direct experimental comparison, their relative affinities are unifi...

Biogenesis of mitochondrial cytochrome c oxidase (COX) is a complex process involving the coordinate expression and assembly of numerous subunits (SU) of dual genetic origin. Moreover, several auxiliary factors are required to recruit and insert the redox-active metal compounds, which in most cases are buried in their protein scaffold deep inside the membrane. Here we used a combination of gel electrophoresis and pull-down assay techniques in conjunction with immunostaining as well as complexome profiling to identify and analyze the composition of assembly intermediates in solubilized membranes of the bacterium Paracoccus denitrificans. Our results show that the central SUI passes through at least three intermediate complexes with distinct subunit and cofactor composition before formation of the holoenzyme and its subsequent integration into supercomplexes. We propose a model for COX biogenesis in which maturation of newly translated COX SUI is initially assisted by CtaG, a chaperone implicated in CuB site metallation, followed by the interaction with the heme chaperone Surf1c to populate the redox-active metal-heme centers in SUI. Only then the remaining smaller subunits are recruited to form the mature enzyme which ultimately associates with respiratory complexes I and III into supercomplexes.

Objective: To assess the safety, tolerability, and effectiveness of the intravenous immunoglobulin (IVIG) Intratect 100 g/L in a prospective, large-scale non-interventional study (NIS) of patients with a wide range of antibody deficiencies as well as other indications for IVIG, risk factors, and frequency of pre-treatments. Materials and methods: Patients were enrolled at 53 practices and clinics in Germany. After recording of baseline information, each patient was treated according to need, as judged by the physician and guided by the SmPC. Relevant data were acquired from medical records, and the patients completed questionnaires to assess treatment satisfaction and quality of life (QoL). Results: At cut-off for this interim analysis, 488 patients were enrolled (planned: 1,000). 47% were male, age 16 – 91 (median 61) years, with treatment durations up to 2,225 (median 282) days. Indications were primary (32%) and secondary (61%) immunodeficiencies, immune thrombocytopenia (4%), and others (3%). More than 92% of physicians recorded very good effectiveness and satisfaction. Patient satisfaction and QoL increased with time from baseline. Initially, 31% of the SID patients had inadequate IgG trough levels (< 4 g/L), including patients with (37%) and without (63%) previous IVIG treatment. Despite a relatively low IVIG dose (median 0.2 g/kg), trough levels improved: after 3 infusions, only 22% of patients had trough levels < 4 g/L, with a plateau below 17% after 6 infusions. Adverse reactions were observed at a rate of 3% per infusion, whereas 0.08% accounted for serious reactions. Conclusion: Effectiveness, safety, patient satisfaction, and QoL were good, confirming the positive benefit-risk profile of the IVIG.

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