The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional 2 equiv of Zn(II) was added to the as-purified PfMetAP-II, no activity could be detected. The combination of these data with previously reported whole cell studies on EcMetAP-I further supports the suggestion that the in vivo metal ion for all MetAP's is Fe(II). Both Co(II)- and Fe(II)-loaded PfMetAP-II showed similar substrate specificities to EcMetAP-I. Substrate binding was largely affected by the amino acid in the P1 position and the length of the polypeptide. The substrates MSSHRWDW and MP-p-NA showed the smallest Km values while the substrates MGMM and MP-p-NA provided the highest turnover. The catalytic efficiency (kcat/Km) of PfMetAP-II for MP-p-NA at 30 °C was 799 500 and 340 930 M-1 s-1 for Co(II)- and Fe(II)-loaded PfMetAP-II, respectively. Maximum catalytic activity was obtained with 1 equiv of Co(II) or Fe(II), and the dissociation constants (Kd) for the first metal binding site were found to be 50 ± 15 and 20 ± 15 nM for Co(II)- and Fe(II)-substituted PfMetAP-II, respectively. Electronic absorption spectral titration of a 1 mM sample of apo-PfMetAP-II with Co(II) provided a dissociation constant of 0.35 ± 0.02 mM for the second metal binding site, a 17500-fold increase compared to the first metal binding site. The electronic absorption data also indicated that both Co(II) ions reside in a pentacoordinate geometry. PfMetAP-II shows unique thermostability and the optimal temperature for substrate turnover was found to be ∼85 °C at pH 7.5 in 25 mM Hepes and 150 mM KCl buffer. The hydrolysis of MGMM was measured in triplicate between 25 and 85 °C at eight substrate concentrations ranging from 2 to 20 mM. Both specific activity and Km values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25−85 °C, indicating that the rate-limiting step in PfMetAP-II peptide hydrolysis does not change as a function of temperature. Co(II)- and Fe(II)-loaded PfMetAP-II have similar activation energies (13.3 and 19.4 kJ/mol, respectively). The thermodynamic parameters calculated at 25 °C are as follows:  ΔG⧧ = 46.23 kJ/mol, ΔH⧧ = 10.79 kJ/mol, and ΔS⧧ = −119.72 J·mol-1·K-1 for Co(II)-loaded PfMetAP; ΔG⧧ = 46.44 kJ/mol, ΔH⧧ = 16.94 kJ/mol, and ΔS⧧ = −99.67 J·mol-1·K-1 for Fe(II)-loaded PfMetAP. Interestingly, at higher temperatures (>50 °C), Fe(II)-loaded PfMetAP-II is more active (1.4-fold at 85 °C) than Co(II)-loaded PfMetAP-II.