Crystal Structure of a Novel Red Copper Protein from Nitrosomonas europaea^,

Abstract

Nitrosocyanin (NC) is a mononuclear red copper protein isolated from the ammonia oxidizing bacterium Nitrosomonas europaea. Although NC exhibits some sequence homology to classic blue copper proteins, its spectroscopic and electrochemical properties are drastically different. The 1.65 A resolution crystal structure of oxidized NC reveals an unprecedented trimer of single domain cupredoxins. Each copper center is partially covered by an unusual extended beta-hairpin structure from an adjacent monomer. The copper ion is coordinated by His 98, His 103, Cys 95, a single side chain oxygen of Glu 60, and a solvent molecule. In the 2.3 A resolution structure of reduced NC, His 98 shifts away from the copper ion, and the solvent molecule is not observed. The arrangement of these ligands renders the coordination geometry of the NC red copper center distinct from that of blue copper centers. In particular, the red copper center has a higher coordination number and lacks the long Cu-S(Met) and short Cu-S(Cys) bond distances characteristic of blue copper. Moreover, the red copper center is square pyramidal whereas blue copper is typically distorted tetrahedral. Analysis of the NC structure provides insight into possible functions of this new type of biological copper center.

Human Sco1 and Sco2 Function as Copper-binding Proteins

“…2A). The visible absorption bands resembled the S-Cu(II) charge transfer bands of nitrosocyanin (30,31), and the presence of bound Cu(II) in human Sco1 as purified was confirmed by EPR (Fig. 3A).…”

“…The Cu(II) site that yields the S-Cu(II) charge transfer transitions in the visible spectral region is likely to have additional donor atoms. The red Cu(II) protein nitrosocyanin binds Cu(II) in a square pyramidal geometry using two N(His) ligands, and single S(Cys), O(Glu), and solvent ligands (30). The visible transitions arise from S(Cys)-to-Cu charge-transfer transitions dominated by rather than donor interactions (31).…”

Metal-binding loop length and not sequence dictates structure

“…All of the noncovalent bonds that stabilize the monomer are still present except in the hinge region, which is the mutated loop. A strand-swapped dimer arrangement has not been seen for a cupredoxin, but in nitrosocyanin an extended ␤-hairpin between the first 2 strands is involved in trimer formation (40). It thus appears that the loop in AZ3A3A, which only differs in length by 1 residue to that of the WT protein and AZ4A3A, alters stability and favors the formation of a strand-swapped dimer, rather than the desired monomeric ␤-barrel.…”

Characterization of a Nitrite Reductase Involved in Nitrifier Denitrification

“…The NcgB and NcgC proteins, also encoded by the nirK operon, are obvious candidates, but these two proteins form a complex, which is not typical of small electron carrier proteins. Other possible candidates include the tetraheme cytochrome c 554 (the electron carrier from hydroxylamine oxidoreductase to the quinone oxidase), cytochrome c 552 (cytochrome c analog), and nitrosocyanin (54,55).…”