The refined structure of functional unit h of keyhole limpet hemocyanin (KLH1-h) reveals disulfide bridges.

Hemocyanins are multimeric oxygen-transport proteins in the hemolymph of many arthropods and mollusks. The overall molecular architecture of arthropod and molluscan hemocyanin is very different, although they possess a similar binuclear type 3 copper center to bind oxygen in a side-on conformation. Gastropod hemocyanin is a 35 nm cylindrical didecamer (2 × 10-mer) based on a 400 kDa subunit. The latter is subdivided into eight paralogous "functional units" (FU-a to FU-h), each with an active site. FU-a to FU-f contribute to the cylinder wall, whereas FU-g and FU-h form the internal collar complex. Atomic structures of FU-e and FU-g, and a 9 Å cryoEM structure of the 8 MDa didecamer are available. Recently, the structure of keyhole limpet hemocyanin FU-h (KLH1-h) was presented as a C(α) -trace at 4 Å resolution. Unlike the other seven FU types, FU-h contains an additional C-terminal domain with a cupredoxin-like fold. Because of the resolution limit of 4 Å, in some loops, the course of the protein backbone could not be established with high certainty yet. Here, we present a refined atomic structure of FU-h (KLH1-h) obtained from low-resolution refinement, which unambiguously establishes the course of the polypeptide backbone and reveals the disulfide bridges as well as the orientation of bulky amino acids.