Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli

The active site of heme catalases is buried deep inside a structurally high ly conserved homotetramer. Channels leading to the active site have been id entified as potential routes for substrate flow and product release, althou gh evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at similar to2.0-Angstrom resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the he me distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the h eme distal pocket filling the main channel. The differences in continuity o f solvent molecules between the native and variant structures illustrate ho w sensitive the solvent matrix is to subtle changes in structure. It is hyp othesized that the slightly larger H2O2 passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent a nd peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-Angstrom resolution, revealing th e existence of hydrogen peroxide binding sites both in the heme distal pock et and in the main channel. Unexpectedly, the largest changes in protein st ructure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departu re from the 222-point group symmetry of the native enzyme. An active role f or channels in the selective flow of substrates through the catalase molecu le is proposed as an integral feature of the catalytic mechanism. The Asn20 1His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathway s of the two reactions can be uncoupled. (C) 2001 Wiley-Liss, Inc.