Human Ceruloplasmin

Abstract

Pulse radiolytic reduction of disulfide bridges in ceruloplasmin yielding RSSR ؊ radicals induces a cascade of intramolecular electron transfer (ET) processes. Based on the three-dimensional structure of ceruloplasmin identification of individual kinetically active disulfide groups and type 1 (T1) copper centers, the following is proposed. The first T1 copper(II) ion to be reduced in ceruloplasmin is the blue copper center of domain 6 (T1A) by ET from RSSR ؊ of domain 5. The rate constant is 28 ؎ 2 s ؊1 at 279 K and pH 7.0. T1A is in close covalent contact with the type 3 copper pair and indeed electron equilibration between T1A and the trinuclear copper center in the domain 1-6 interface takes place with a rate constant of 2.9 ؎ 0.6 s ؊1 . The equilibrium constant is 0.17. Following reduction of T1A Cu(II), another ET process takes place between RSSR ؊ and T1B copper(II) of domain 4 with a rate constant of 3.9 ؎ 0.8. No reoxidation of T1B Cu(I) could be resolved. It appears that the third T1 center (T1C of domain 2) is not participating in intramolecular ET, as it seems to be in a reduced state in the resting enzyme.The blue multicopper oxidases are enzymes that catalyze the 4-electron reduction of dioxygen to water by four sequential 1-electron oxidations of substrate (1). These enzymes are widely distributed in nature, from bacteria, fungi, and plants to mammals. All contain at least 4 copper ions of the following types. (i) The blue type 1 site (T1) 1 characterized by an intense charge transfer band in the 600-nm region (⑀ ϳ 5000 M Ϫ1 cm Ϫ1 ) and a narrow hyperfine coupling constant (A ʈ Ͻ 10 Ϫ3 cm Ϫ1 ) in the electron paramagnetic resonance spectrum (2). (ii) A "normal" type 2 (T2) copper center characterized by a lack of intense absorption bands and ordinary EPR spectrum. (iii) A copper ion pair, called type 3 (T3), which in the oxidized state is characterized by an intense absorption in the near UV region (⑀ ϳ 4000 M Ϫ1 cm Ϫ1 ) and by strong antiferromagnetic coupling. T2 and T3 are proximal and form together a trinuclear cluster, which is the dioxygen reduction site (1). The physiological function of T1 is sequential uptake and transfer of single electrons from substrate molecules to the trinuclear center where dioxygen binds and gets reduced to water. Thus, the enzymatic process takes place by a ping-pong mechanism (1).Intramolecular electron transfer (ET) between T1 and the trinuclear T2/T3 center is therefore expected to play a crucial role in the molecular mechanism of this class of enzymes. High resolution crystal structures are now available for ascorbate oxidase (3, 4), ceruloplasmin (5, 6), and fungal laccase (7). The laccases are characterized by relatively low substrate specificity toward the reducing substrate, whereas ascorbate oxidase exhibits a high specificity toward L-ascorbate. We have previously studied the intramolecular ET processes that take place in Rhus vernicifera laccase (8) and in ascorbate oxidase (9 -11).The only blue copper oxidase found in humans is ceruloplasmin (hCp) (Fe(I...

Site-directed Mutagenesis of Human Ceruloplasmin

“…These latter sites have redox potential values higher than those of typical T1 sites. Whether and how all three T1 sites take part in the turnover reaction of Cp is presently a major question (27). Only the T1 site of domain 6 is connected by the cysteinehistidine linkages to the trinuclear cluster (23).…”

“…With the exception of Cp, there is generally one T1 site per trinuclear cluster in these enzymes. A conserved structural motif of the polypeptide chain, His-Cys His, where the Cys is a ligand of the T1 site and the two His residues are ligands of two copper atoms of the cluster, facilitates the intramolecular electron transfer (ET) between the two sites, ϳ13 Å apart (26,27).…”

“…Fet3 has only one T1 site (29) and is as able as Cp to oxidize both Fe(II) ions and amines. Structural and catalytic studies are trying to find plausible electron transfer pathways in the molecule to disclose the functional relations of the three sites, if any (27), and their interaction with the cluster during catalysis (15).…”

Electron Transfer Reactivity of the Arabidopsis thaliana Sulfhydryl Oxidase AtErv1

“…In addition to the advantageous time resolution of this method, it enables production of reagents with a wide range of reduction potentials (22). A disulfide bridge has been used previously as an entry port for pulse radiolytically produced reduction equivalents in studies of electron transfer through proteins including azurin (23) and ceruloplasmin (24). An elegant example of thorough analysis by pulse radiolysis of a flavin-containing enzyme is the work of Hille on xanthine oxidase (25).…”

Elucidation of Electron‐ Transfer Pathways in Copper and Iron Proteins by Pulse Radiolysis Experiments

“…The CO 2 À radicals react with disulfide groups in hCp at diffusion controlled rates to produce the RSSR À radicals monitored at 410 nm (where RSSR À radicals exhibit an absorption maximum) while no direct reduction of T1Cu II was observed (72). Instead, the electrons were further transferred to a T1(Cu II ) center in an intramolecular process with a rate constant of 28 AE 2 s À1 at 279 K. An intramolecular electron equilibration with the T2/T3 center was then observed with a rate constant of 2:9 AE 0:6 s À1 determined independently at both 610 nm (T1Cu II absorption maximum) and at 330 nm, where the oxidation state of the trinuclear center can be monitored independently [ Fig.…”