The thermodynamics, kinetics, and molecular mechanism of intramolecular electron transfer in human ceruloplasmin

Ceruloplasmin is a multicopper oxidase that contains three type 1 Cu sites and a type 2/type 3 trinuclear Cu cluster. All other multicopper oxidases c ontain only one type 1 Cu and a trinuclear cluster. In oxidized human cerul oplasmin, one type 1 Cu site is reduced and cannot be oxidized, at least in part due at to a high reduction potential, which is not catalytically rele vant. Here we have examined the thermodynamics and kinetics of electron tra nsfer among the five redox-active Cu sites to obtain insight into the molec ular mechanism of intramolecular electron transfer and the function of the additional, redox-active T1 Cu site. The redox potentials of the Cu sites o f human ceruloplasmin were determined by reductive and poised potential tit rations. In pH 7.0 phosphate buffer, the potentials of the type 1, type 2. and type 3 Cu sites are 448, 491, and 415 mV, respectively. Cl- binds to th e trinuclear cluster and significantly increases the potentials of the type 2 and type 3 Cu, indicating that Cl- is a physiologically relevant effecto r of the redox potentials of the Cu sites. Reductive titrations monitored b y EPR indicate that the redox potentials of the two redox-active type 1 Cu sites are the same. Upon reduction and reoxidation with O-2, the trinuclear cluster and 50% of the redox-active type 1 Cu are reoxidized. From EPR, th is additional electron is distributed equally between the two redox-active type 1 Cu sites. Rapid freeze-quench EPR demonstrated that the rate of elec tron transfer between the two T1 Cu sites is >150 s(-1), which is faster th an the rate of decay of the native intermediate state and indicates that bo th sites may be catalytically relevant. After reoxidation, the additional e lectron does not transfer to the trinuclear cluster. Addition of 1-2 equiv of Fe(II) induces ET to the type 2 and T3 Cu sites, indicating that the tri nuclear cluster requires at least two electrons to be reduced. Kinetics of reduction of the oxidized enzyme were also studied. Reduction of the type 1 and type 3 Cu sites is fast, while reduction of the type 2 Cu site is slow , indicating that the type 3 Cu pair is reduced by a reduced type I Cu and another Fe(II). These results provide new insight into the molecular mechan ism of intramolecular electron transfer in ceruloplasmin. Possible electron -transfer pathways among the Cu sites were examined, and the role of two fu nctional type 1 Cu sites is discussed.