CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich alpha-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase.

The mechanism of the four-electron reduction of dioxygen by a multicopper oxidase, CueO, was studied based on reactions of single and double mutants with Cys⁵⁰⁰, a type I copper ligand, and the noncoordinating Asp¹¹² and Glu⁵⁰⁶, which form hydrogen bonds with the trinuclear copper center directly and indirectly via a water molecule. The reaction of C500S containing a vacant type I copper center produced intermediate I in an EPR-silent peroxide-bound form. The formation of intermediate I from C500S/D112N was restricted due to a reduction in the affinity of the trinuclear copper center for dioxygen. The state of intermediate I was realized to be the resting form of C500S/E506Q and C500S of the truncated mutant Δα5–7CueO, in which the 50 amino acids covering the substrate-binding site were removed. Reactions of the recombinant CueO and E506Q afforded intermediate II, a fully oxidized form different from the resting one, with a very broad EPR signal, g < 2, detectable only at cryogenic temperatures and unsaturated with high power microwaves. The lifetime of intermediate II was prolonged by the mutation at Glu⁵⁰⁶ involved in the donation of protons. The structure of intermediates I and II and the mechanism of the four-electron reduction of dioxygen driven by Asp¹¹² and Glu⁵⁰⁶ are discussed.

From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum ), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c ₅₅₄ . These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c ₅₅₁ , composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c ₅₅₄ reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae , etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.

Bacillus subtilis yumC encodes a novel type of ferredoxin‐NADP⁺ oxidoreductase (FNR) with a primary sequence and oligomeric conformation distinct from those of previously known FNRs. In this study, the crystal structure of B. subtilis FNR (BsFNR) complexed with NADP⁺ has been determined. BsFNR features two distinct binding domains for FAD and NADPH in accordance with its structural similarity to Escherichia coli NADPH‐thioredoxin reductase (TdR) and TdR‐like protein from Thermus thermophilus HB8 (PDB code: 2ZBW). The deduced mode of NADP⁺ binding to the BsFNR molecule is nonproductive in that the nicotinamide and isoalloxazine rings are over 15 Å apart. A unique C‐terminal extension, not found in E. coli TdR but in TdR‐like protein from T. thermophilus HB8, covers the re‐face of the isoalloxazine moiety of FAD. In particular, Tyr50 in the FAD‐binding region and His324 in the C‐terminal extension stack on the si‐ and re‐faces of the isoalloxazine ring of FAD, respectively. Aromatic residues corresponding to Tyr50 and His324 are also found in the plastid‐type FNR superfamily of enzymes, and the residue corresponding to His324 has been reported to be responsible for nucleotide specificity. In contrast to the plastid‐type FNRs, replacement of His324 with Phe or Ser had little effect on the specificity or reactivity of BsFNR with NAD(P)H, whereas replacement of Arg190, which interacts with the 2′‐phosphate of NADP⁺, drastically decreased its affinity toward NADPH. This implies that BsFNR adopts the same nucleotide binding mode as the TdR enzyme family and that aromatic residue on the re‐face of FAD is hardly relevant to the nucleotide selectivity.