Identification of a Blue Copper Protein from Hyphomicrobium denitrificans and its Functions in the Periplasm

Abstract

It has been known that the methylotrophic denitrifying bacteria have the specific electron transfer chains, involving in 'methanol oxidation' and 'denitrification', in the periplasm. Recently, a unique blue copper protein (HdBCP) has been isolated from the methanol-grown methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans. HdBCP is a 14.5 kDa protein and contains one copper atom in the molecule. The electronic absorption spectrum of HdBCP exhibits two absorption maxima near 450 and 750 nm comparable with the intense 600 nm band (epsilon(450)/epsilon(600) = ca. 0.9). The rhombic electron paramagnetic resonance spectrum shows clearly that the copper centre is a 'perturbed' type 1 copper geometry. Stopped-flow kinetics indicates that HdBCP accepts efficiently an electron from cytochrome c(L) (k(2) = 4.0 x 10(6) M(-1) s(-1) at 25.0 degrees C), which is a physiological electron acceptor for methanol dehydrogenase. According to cloning and DNA sequencing of the structural gene, the deduced amino acid sequence shows significant similarities with pseudoazurins, which are a physiological electron donor for Cu-containing nitrite reductase from the denitrifying bacteria. Based on these results, we discuss the role of HdBCP in the electron-flow system, which link 'methanol oxidation' and 'denitrification' together.