Crystallographic structures of bovine copper-zinc superoxide dismutase reveal asymmetry in two subunits: functionally important three and five coordinate copper sites captured in the same crystal

A key feature of the generally accepted catalytic mechanism of CuZn superoxide dismutase (CuZnSOD) is the breakage of the imidazolate bridge between copper and zinc and the loss of a coordinated water molecule from copper on reduction from Cu(II) to Cu(I). Crystal structures exist for the enzyme from a number of sources in the oxidised, five coordinate copper form. For the reduced form two structures from different sources have been determined only recently but provide contradictory results. We present crystal structures of bovine CuZnSOD (BSOD) in two different space groups. The structure of the P212121 form (pBSOD), at 1.65 A resolution clearly shows one subunit with Cu in the five coordinate, oxidised form, and the other with Cu in the three coordinate form expected for the reduced state. This mixed state of pBSOD is confirmed by XANES data of these crystals. The pBSOD structure has thus captured each subunit in one of the two oxidation state conformations and thus provides direct crystallographic evidence for the superoxide dismutase mechanism involving the breakage of the imidazole bridge between Cu and Zn. A shift in the position of copper in subunit A poises the catalytic centre to undergo the first stage of catalysis via dissociation of Cu from His61 with a concomittant movement of the coordinated water molecule towards His61, which rotates by approximately 20 degrees, enabling it to form a hydrogen bond to the water molecule. The Cu-Zn separation in the reduced site is increased by approximately 0.5 A. In contrast the 2.3 A resolution structure in space group C2221 (cBSOD) shows both of the Cu atoms to be in the five coordinate, oxidised form but in this space group the whole of subunit A is significantly more disordered than subunit B. An examination of published structures of "oxidised" SODs, shows a trend towards longer Cu-Zn and Cu-His61 separations in subunit A, which together with the structures reported here indicate a potential functional asymmetry between the subunits of CuZnSODs. We also suggest that the increased separation between Cu and Zn is a precursor to breakage of His61.     

Conformational variability of the Cu site in one subunit of bovine CuZn superoxide dismutase: the importance of mobility in the Glu119-Leu142 loop region for catalytic function

The structure of the catalytic site in one subunit of bovine CuZn superoxide dismutase is shown to be highly variable. A series of crystal structures at approximately 1.7 A have been determined using data collected from different crystals. Several conformations are observed for the copper site from one of the subunits. These conformations lie between those expected for the Cu(II) and Cu(I) forms of the enzyme and may represent a slow positional rearrangement of the Cu site during the crystallisation process due to the presence of a trace reductant in the mother liquor. These states perhaps indicate some functionally relevant structural steps that ultimately result in the breakage of the imidazolate bridge between the two metal sites.This behaviour is not observed for the second subunit of the dimeric enzyme, which remains in the five-coordinate, distorted square planar geometry in all cases. We suggest that this asymmetric behaviour may be caused by the lack of mobility for the Glu119-Leu142 loop region in the second subunit caused by crystal contacts. This region forms one wall of the active-site cavity, and its mobility has been suggested, via molecular dynamics studies, to be important for the catalytic mechanism.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Redox-dependent structural changes in an engineered heme-copper center in myoglobin: insights into chloride binding to CuB in heme copper oxidases

The effects of chloride on the redox properties of an engineered binuclear heme-copper center in myoglobin (Cu(B)Mb) were studied by UV-vis spectroelectrochemistry and EPR spectroscopy. A low-spin heme Fe(III)-Cu(I) intermediate was observed during the redox titration of Cu(B)Mb only in the presence of both Cu(II) and chloride. Upon the first electron transfer to the Cu(B) center, one of the His ligands of Cu(B) center dissociates and coordinates to the heme iron, forming a six-coordinate low-spin ferric heme center and a reduced Cu(B) center. The second electron transfer reduces the ferric heme and causes the release of the coordinated His ligand. Thus, the fully reduced state of the heme-copper center contains a five-coordinate ferrous heme and a reduced Cu(B) center, ready for O(2) binding and reduction to water to occur. In the absence of a chloride ion, formation of the low-spin heme species was not observed. These redox reactions are completely reversible. These results indicate that binding of chloride to the Cu(B) center can induce redox-dependent structural changes, and the bound chloride and hydroxide in the heme-copper center may play different roles in the redox-linked enzymatic reactions of heme-copper oxidases, probably because of their different binding affinity to the copper center and the relatively high concentration of chloride under physiological conditions.     

Subunit asymmetry in the three-dimensional structure of a human CuZnSOD mutant found in familial amyotrophic lateral sclerosis.

The X-ray crystal structure of a human copper/zinc superoxide dismutase mutant (G37R CuZnSOD) found in some patients with the inherited form of Lou Gehrig's disease (FALS) has been determined to 1.9 angstroms resolution. The two SOD subunits have distinct environments in the crystal and are different in structure at their copper binding sites. One subunit (subunit[intact]) shows a four-coordinate ligand geometry of the copper ion, whereas the other subunit (subunit[broken]) shows a three-coordinate geometry of the copper ion. Also, subunit(intact) displays higher atomic displacement parameters for backbone atoms ((B) = 30 +/- 10 angstroms2) than subunit(broken) ((B) = 24 +/- 11 angstroms2). This structure is the first CuZnSOD to show large differences between the two subunits. Factors that may contribute to these differences are discussed and a possible link of a looser structure to FALS is suggested.     

Complexation of the antihypertensive drug oxprenolol with copper(II)

The complexation between copper(II) and the antihypertensive drug oxprenolol (HOxp) was studied both in methanol and slightly alkaline aqueous media at Cu:HOxp molar ratio from 1:1 to 1:10. Copper(lI) forms two types of complexes-a mononuclear violet one, CuOxp2, with bidentately bound ligands and a green dimeric one, Cu2Oxp2Cl2, in which the two Cu(II) centres are linked by the ligand through oxygen bridges. The crystal structure of the Cu2Oxp2Cl2 complex consists of two crystallographically non-equivalent centrosymmetric copper dimers. Each copper atom is four-coordinated in a distorted square-planar environment. The Cu2O2 structural core is characterized by a Cu1-O1-Cu1' angle of 104.15(13)degrees (Cu2-O2-Cu2' 104.30(13) degrees) and a relatively short Cu1-Cu1' separation of 3.026(1) A (Cu2-Cu2'-3.023(1) A). Magnetic susceptibility and EPR measurements indicate an antiferromagnetic coupling of the copper(II) centers.     

Ab initio modelling of the structure and redox behaviour of copper(I) bound to a His–His model peptide: relevance to the β-amyloid peptide of Alzheimer’s disease

A contributing factor to the pathology of Alzheimer's disease is the generation of reactive oxygen species, most probably a consequence of the beta-amyloid (Abeta) peptide coordinating copper ions. Experimental and theoretical results indicate that His13 and His14 are the two most firmly established ligands in the coordination sphere of Cu(II) bound to Abeta. Abeta1-42 is known to reduce Cu(II) to Cu(I). The Abeta-Cu(II) complex has been shown to catalytically generate H(2)O(2) from reducing agents and O(2). Cu(II) in the presence of Abeta has been reported to have a formal reduction potential of +0.72-0.77 V (vs. the standard hydrogen electrode). Quantum chemical calculations using the B3LYP hybrid density functional method with the 6-31G(d) basis set were performed to model the reduction of previously studied Cu(II) complexes representing the His13-His14 portion of Abeta (Raffa et al. in J. Biol. Inorg. Chem. 10:887-902, 2005). The effects of solvation were accommodated using the CPCM method. The most stable complex between Cu(I) and the model compound, 3-(5-imidazolyl)propionylhistamine (1) involves tricoordinated Cu(I) in a distorted-T geometry, with the Npi of both imidazoles as well as the oxygen of the backbone carbonyl bound to copper. This model would be the most likely representation of a Cu(I) binding site for a His-His peptide in aqueous solution. A variety of possible redox processes are discussed.     

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.     

Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties

Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (相似文献   

Kinetics of the oxidation of reduced Cu,Zn-superoxide dismutase by peroxymonocarbonate

Kinetic evidence is reported for the role of the peroxymonocarbonate, HOOCO(2)(-), as an oxidant for reduced Cu,Zn-superoxide dismutase-Cu(I) (SOD1) during the peroxidase activity of the enzyme. The formation of this reactive oxygen species results from the equilibrium between hydrogen peroxide and bicarbonate. Recently, peroxymonocarbonate has been proposed to be a key substrate for reduced SOD1 and has been shown to oxidize SOD1-Cu(I) to SOD1-Cu(II) much faster than H(2)O(2). We have reinvestigated the kinetics of the reaction between SOD1-Cu(I) and HOOCO(2)(-) by using conventional stopped-flow spectrophotometry and obtained a second-order rate constant of k=1600±100M(-1)s(-1) for SOD1-Cu(I) oxidation by HOOCO(2)(-). Our results demonstrate that peroxymonocarbonate oxidizes SOD1-Cu(I) to SOD1-Cu(II) and is in turn reduced to the carbonate anion radical. It is proposed that the dissociation of His61 from the active site Cu(I) in SOD-Cu(I) contributes to this chemistry by facilitating the binding of larger anions, such as peroxymonocarbonate.     

Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity

Oxidative DNA damage caused by a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and a cysteine as an electron donor was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Unlike CuZnSOD, manganese SOD (MnSOD) as well as iron SOD (FeSOD) did not enhance DNA damage. The capacity of CuZnSOD to enhance damage to DNA was inhibited by a spin-trapping agent, 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) and a metal chelator, diethylenetriaminepentaacetic acid (DETAPAC). The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with Cys-MCO. We found that the Cys-MCO system caused the release of free copper from CuZnSOD. CuZnSOD also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on Cys-MCO-induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged CuZnSOD, and H(2)O(2) produced by the Cys-MCO system.     

Structure of fully reduced bovine copper zinc superoxide dismutase at 1.15 A

Copper zinc superoxide dismutase (CuZnSOD) forms a crucial component of the cellular response to oxidative stress by catalyzing the dismutation of the superoxide radical to hydrogen peroxide and water. Mutations in human CuZnSOD are associated with the development of familial amyotrophic lateral sclerosis (motor neuron disease). We have determined the structure of fully reduced bovine CuZnSOD to 1.15 A, the only atomic resolution structure for an intact CuZnSOD and one of only a small number for metalloproteins. For the first time, both subunits have been captured with the three coordinate Cu(I) ligation required by the generally accepted catalytic mechanism, where dismutation of the superoxide radical occurs via reduction of Cu. Furthermore, the improved resolution compared to previous studies (to 1.65 A) has allowed a more detailed examination of the metal center environment and its associated water network in the active site channel, facilitating the analysis of potential proton transfer routes.     

Effect of superoxide dismutase deficiency on the life span of the yeast Saccharomyces cerevisiae. An oxygen-independent role of Cu,Zn-superoxide dismutase

Effects of the absence of Cu,Zn-superoxide dismutase (CuZnSOD) on the replicative life span of the yeast Saccharomyces cerevisiae were studied under different oxygen conditions. In both strains, replicative life span and the rate of cell divisions were found to be similar under the atmosphere of air and under hypoxic (3% oxygen) and anoxic conditions. These results indicate that deleterious consequences of the lack of CuZnSOD are not limited to elevation of superoxide concentration and involve function(s) other than superoxide scavenging.     

Electron addition to the active site of Cancer magister haemocyanins. An ESR study of cu(II) centres after gamma-radiolysis.

Exposure of aqueous glasses of Cancer magister haemocyanin to 60Co gamma-rays at 77 K results in a novel paramagnetic centre with ESR features showing hyperfine coupling to one strongly coupled 63/65 Cu nucleus and possibly one weakly interacting 63/65 Cu nucleus. Addition of electron scavengers showed that this centre is formed by electron addition. It is suggested that addition occurs at Cu2+-O2-Cu2+ units to give Cu2+-O2Cu+ centres. If this is correct, then electron-transfer between the two copper ions is slow or non-existent, possibly indicating that they are inequivalent. The centre is unstable, the signals being lost irreversibly on heating to approx. 270 K. The g parallel and A parallel (63/65Cu) data place this centre into the type I classification.     

Crystallographic determination of reduced bovine superoxide dismutase at pH 5.0 and of anion binding to its active site

The crystal structures of dithionite-reduced bovine Cu(I),Zn superoxide dismutase and of its adducts with the inorganic anions azide and thyocyanide have been determined in a C222₁ crystal form obtained at pH?5.0. This crystal form is characterized by a high solvent content (72%) and by having the two Cu,ZnSOD monomers (A and B) in different crystal environments. One of them (B) is involved in few intermolecular crystal contacts so that it is in a more "solution like" environment, as indicated by average temperature factors which are about twice those of the other monomer. The differences in crystal packing affect the active site structures. While in the A monomer the Cu(I) is coordinated to all four histidine residues, in the B monomer the bridging His61 side chain is found disordered, implying partial detachment from copper. The same effect occurs in the structures of the anion complexes. The inorganic anions are found bound in the active site cavity, weakly interacting with copper at distances ranging from 2.5 to 2.8?Å. The copper site in the A subunit of the native enzyme structure displays significant electron density resembling a diatomic molecule, bound side-on at about 2.8?Å from the metal, which cannot be unambiguously interpreted. The crystallographic data suggest that the existence of the His61 bridge between copper and zinc is dominated by steric more than electronic factors and that the solution state favors the His61 detachment. These structures confirm the existence of an energetically available state for Cu(I) in Cu,ZnSOD where the histidinato bridge to zinc is maintained. This state appears to be favored by tighter crystal contacts. The binding of the anions in the active site cavity is different from that observed in the oxidized enzyme and it appears to be dominated by electrostatic interactions within the cavity. The anion binding mode observed may model the substrate interaction with the reduced enzyme during catalysis.     

Effect of dietary copper deficiency on the distribution of dopamine and norepinephrine in mice and rats

Dietary copper deficiency was produced in Swiss albino mice and Sprague Dawley rats to determine the organ specificity of alterations in norepinephrine (NE) and dopamine (DA) concentrations and the relationship with organ copper levels. A 5-week dietary treatment was used, which started 1 week after birth for mice, initially via dams, and 3 weeks after birth for rats. Mice offspring (6 weeks of age) and rats (8 weeks of age) maintained on a copper-deficient (-Cu) treatment were compared with copper-adequate (+Cu) controls. Compared with +Cu animals, -Cu mice and rats were anemic and had low (<1% of +Cu) ceruloplasmin activities but normal body weights. The -Cu mice had organ copper concentrations ranging between 30% and 65% of +Cu values for eight organs studied, with the thymus being the least depleted. For -Cu rats, the range was 15% to 65%. Significant reductions in NE concentration were observed in the heart, pancreas, and spleen of -Cu mice. Elevated DA levels were observed in all organs except the brain. For -Cu rats, the NE level was lower in the heart and the DA level was higher in both the heart and spleen compared with +Cu rats. Dopamine elevation in the heart and spleen for both -Cu mice and rats was four- and fivefold higher, respectively. Adrenal catecholamine levels were only slightly changed by copper deficiency in mice or rats. Urinary levels of both NE and DA were higher in -Cu rats and mice. Plasma and heart tyrosine levels were not altered in -Cu mice. Elevated DA in -Cu rodents may be due to limiting dopamine-beta-monooxygenase. Higher urinary NE and lower organ NE may be due to a combination of decreased synthesis and enhanced turnover. The magnitude of decreased organ copper was not predictive of altered catecholamine pool size.     

The Cu(II) acetate complex of the heteroscorpionate ligand (2-hydroxy-3-t-butyl-methylphenyl)bis(3,5-dimethylpyrazolyl)methane: a structural model for copper-substituted serralysin and astacin

Reaction of Cu(II) acetate with the deprotonated ligand (2-hydroxy-3-t-butyl-methylphenyl)bis(3,5-dimethylpyrazolyl)methane (L1O(-)) in methanol produced the complexes [(L1O)Cu(OAc)], a=9.275(2), b=11.641(5), c=13.532(3) A, alpha=69.62(1) degrees, beta=89.49(2) degrees, gamma=87.12(1) degrees, P1. The Cu adopts a distorted five-coordinate geometry where the two pyrazole nitrogen ligands and a bidentate acetate occupy the pseudoequatorial plane with the phenoxy oxygen in an apical position. This complex has a number of features in common with galactose oxidase and the copper-substituted endopeptidases serralysin and astacin.     

Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme.

Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species. One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD). To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C. crescentus CB15 was cloned and sequenced. By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C. crescentus chromosome. Through analysis of spheroplasts, the two SODs of C. crescentus were shown to be differently localized, CuZnSOD in the periplasm and FeSOD in the cytoplasm. In its natural habitat, C. crescentus is frequently associated with blue-green algae (cyanobacteria). The oxygen evolved by these photosynthetic algae may create an extracellular oxidative stress against which the periplasmic CuZnSOD may defend more effectively than the cytoplasmic FeSOD. Amino acid sequence alignments of C. crescentus CuZnSOD with eucaryotic CuZnSODs and with CuZnSOD of Photobacterium leiognathi (the only other bacterium from which CuZnSOD has been isolated and sequenced) suggest similar supersecondary structures for bacterial and eucaryotic CuZnSODs but reveal four novel substitutions in C. crescentus CuZnSOD: a phenylalanine critical to intrasubunit hydrophobic bonding replaced by alanine, a histidine ligand of zinc replaced by aspartate, and substitutions of two other previously invariant residues that stabilize zinc or both copper and zinc. These amino acid substitutions in C. crescentus CuZnSOD may have implications for its catalysis and stability.     

Structural and Electronic Snapshots during the Transition from a Cu(II) to Cu(I) Metal Center of a Lytic Polysaccharide Monooxygenase by X-ray Photoreduction

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.     

Glutathione-mediated transfer of copper(I) into American lobster apohemocyanin.

Copper in the cytosol of the hepatopancreas of the American lobster, Homarus americanus, occurs as copper-metallothionein [Cu(I)-MT] and as a copper-glutathione complex [Cu(I)-GSH]. The latter can act in vitro as the source of Cu(I) in the reconstitution of lobster apohemocyanin, whereas Cu(I)-MT cannot. Here we report on the mechanism of the GSH-mediated reconstitution. Binding of Cu(I) to apohemocyanin was measured by its effect on the protein's fluorescence, by ultrafiltration experiments and size-exclusion HPLC. Reconstitution of CO and O2 binding was studied using the [Cu(I)...Cu(I)-CO] fluorescence of hemocyanin and its Cu-O2-Cu charge-transfer band as spectral probes. The hemocyanin oligomer has 1 (1.02 +/- 0.09) high-affinity (apparent Kdiss = 1.67 +/- 0.40 microM) external binding site for ionic Cu(I) per subunit. Binding of Cu(I) to this site is fast and reversible and is followed by a slow, irreversible incorporation of copper into the protein matrix. Movement of the first copper through the matrix to the active site is the rate-limiting step in the reconstitution process. Mononuclear copper sites, once formed, are rapidly converted into biologically active, binuclear copper sites. In accordance with this reaction sequence, the restoration of CO/O2 binding by hemocyanin is a first-order reaction with a half-time of 100 +/- 5 min at pH 6.0. Reconstitution is extremely pH-dependent and proceeds best at those pH values where the architecture of the copper pocket of hemocyanin is open as judged from its extremely low affinity for oxygen and its very fast oxygen dissociation rate.(ABSTRACT TRUNCATED AT 250 WORDS)