The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 Å resolution. The protein was crystallized using reagents prepared in D 2 O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copperbinding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein.Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-H⋯X bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.copper protein | hydrogen/deuterium exchange | hydrogen bonding | oxidation/reduction | redox potential C upredoxins comprise a class of Type I blue copper proteins that participates in respiratory, anabolic and catabolic processes in plants and bacteria by mediating biological electron transfer (ET) (1, 2). Amicyanin is an 11.5 kDa protein that belongs to a distinct subclass of cupredoxins found in bacteria. It differs from its closest relatives, plastocyanin, and pseudoazurin, by having 20 additional residues at its N-terminus (3). In the copper-binding site of amicyanin, two histidine, one cysteine, and one methionine provide copper ligands. Amicyanin mediates ET from methylamine dehydrogenase (MADH), a quinoprotein containing the tryptophan tryptophylquinone prosthetic group, to c-type cytochromes (4, 5). Several x-ray crystallographic studies have been carried out on amicyanin from Paracoccus denitrificans, its mutants, its binary complex with MADH, and its ternary complex with MADH and cytochrome c-551i (3, 6-11). These x-ray studies in combination with spectroscopic and kinetic studies established this ET complex as a paradigm for elucidating factors that control rates of ET through and between proteins (12). Specific amino acid residues of amicyanin have been altered to modulate the driving force (ΔG o ), electronic coupling (H AB ), or reorganization energy (λ) that in turn effect the ET rates within the MADH-amicyanin-cytochrome c-551i complex (12). Several site-directed mutations that alter the kinetic mechanisms of these ET reactions and convert true ET reactions to coupled or gated ET reactions have also been characterized with this system (12).Previous studies indicated that the electronic properties of am...