To clarify how the location of distal histidine affects the activation process of H 2 O 2 by heme proteins, we have characterized reactions with H 2 O 2 for the L29H/ H64L and F43H/H64L mutants of sperm whale myoglobin (Mb), designed to locate the histidine farther from the heme iron. Whereas the L29H/H64L double substitution retarded the reaction with H 2 O 2 , an 11-fold rate increase versus wild-type Mb was observed for the F43H/ H64L mutant. The V max values for 1-electron oxidations by the myoglobins correlate well with the varied reactivities with H 2 O 2 . The functions of the distal histidine as a general acid-base catalyst were examined based on the reactions with cumene hydroperoxide and cyanide, and only the histidine in F43H/H64L Mb was suggested to facilitate heterolysis of the peroxide bond. The x-ray crystal structures of the mutants confirmed that the distal histidines in F43H/H64L Mb and peroxidase are similar in distance from the heme iron, whereas the distal histidine in L29H/H64L Mb is located too far to enhance heterolysis. Our results indicate that the proper positioning of the distal histidine is essential for the activation of H 2 O 2 by heme enzymes.Peroxidase is a heme enzyme that catalyzes 1-electron oxidations of a variety of substrates (1, 2). The ferric enzyme is oxidized by H 2 O 2 to yield a ferryl porphyrin cation radical (Fe IV ϭO Por . ϩ ) known as compound I in the first step of the catalytic cycle (3). Compound I is reduced to the ferric state through a ferryl species (Fe IV ϭO Por), so-called compound II, by two sequential 1-electron oxidations of substrates. The invariant histidine in the distal heme pocket (1-6) is a critical residue for peroxidases, and its replacement by aliphatic residues retards compound I formation by 5ϳ6 orders of magnitude (7-9). As shown in Scheme I, the distal histidine is believed to function (i) as a general base to accelerate binding of H 2 O 2 to the ferric heme iron by deprotonating the peroxide and (ii) as a general acid to facilitate the heterolytic cleavage of the O-O bond of a plausible Fe III ⅐OOH complex by protonating the terminal oxygen atom (10). The charge separation in heterolysis is suggested to be also enhanced by a positively charged distal arginine (Scheme I), whose substitution results in 2 orders of magnitude slower formation of compound I (10 -12).Myoglobin (Mb), 1 a carrier of molecular oxygen, similarly possesses a distal histidine (His-64) in the heme pocket ( Fig. 1 might be partly due to the malfunction of the distal histidine as a general acid-base catalyst and the absence of the distal arginine. Whereas the distal histidine in peroxidase is suggested to raise the basicity of imidazole by a hydrogen bond with the adjacent asparagine (20, 21), the absence of the hydrogen bond in Mb (13, 14) is indicative of less basicity of its distal histidine. Furthermore, wild-type Mb cleaves the O-O bond of the heme-bound peroxide not only heterolytically, but also homolytically to give Mb-II and a hydroxy radical as shown in Scheme...

Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.

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Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

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