Two types of hetero-oligomeric dye-linked l-proline dehydrogenases (alpha(4)beta(4) and alpha beta gamma delta types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the beta subunit (PDH beta) is responsible for catalyzing l-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDH beta s from alpha(4)beta(4)- and alpha beta gamma delta-type enzyme complexes, eight PDH beta s (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The alpha(4)beta(4)-type PDH beta s showed greater thermostability than most of the alpha beta gamma delta-type PDH beta s: the former retained more than 80 % of their activity after heating at 70 A degrees C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the alpha(4)beta(4)-type PDH beta s utilized ferricyanide as the most preferable electron acceptor, whereas alpha beta gamma delta-type PDH beta s preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDH beta s likely reflect whether they were from an alpha beta gamma delta- or alpha(4)beta(4)-type complex, though the wider divergence observed within alpha beta gamma delta-type PDH beta s based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDH beta s did not reflect the complex type. Interestingly, the k (cat) value for free alpha(4)beta(4)-type PDH beta from P. horikoshii was much larger than the value for the same subunit within the alpha(4)beta(4)-complex. This indicates that the isolated PDH beta could be a useful element for an electrochemical system for detection of l-proline.