HOW REPLACEMENTS OF THE 12 CONSERVED HISTIDINES OF SUBUNIT-I AFFECT ASSEMBLY, COFACTOR BINDING, AND ENZYMATIC-ACTIVITY OF THE BRADYRHIZOBIUM-JAPONICUM CBB(3)-TYPE OXIDASES

Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb(3)-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the follow ing cofactors: the low spin heme B and the binuclear high spin heme B Cu-B center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidin es of FixN in cofactor binding and function of the Bradyrhizobium japo nicum cbb(3)-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Thony-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394 , 349-352). Four of the remaining histidines were not essential for ac tivity or assembly (positions 226, 246, 333, and 457); by contrast, hi stidines 331, 410, and 418 were required both for activity and stabili ty of the enzyme. The last group of mutant enzymes, H420A, H280A, H330 A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was adde d to the C terminus of subunit I. The His(6)-tagged cbb(3)-oxidase com plexes were purified 20-fold by a three-step purification protocol. Wi th the exception of the H420A mutant oxidase, the mutant enzymes H280A , H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics lik e the wild-type His(6)-tagged enzyme. The His(6)-tagged H420A mutant o xidase differed from the His(6)-tagged wild-type protein by showing al tered visible light spectroscopic characteristics. No stable mutant ox idase lacking copper or heme B was obtained. This strongly suggests th at copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.