The dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is an enzyme that converts para-halogenated phenols to the corresponding quinones in the presence of hydrogen peroxide. Its enzymatic activity is similar to that of heme peroxidases such as horseradish peroxidase, yet it has the structural characteristics of the globin family of proteins, the main functions of which are oxygen transport and storage. In order to investigate the dual function of this hemoglobin peroxidase, the enzyme was expressed in Escherichia coli as a recombinant protein in its wild-type form and as a mutant protein in which Cys73 was replaced by a serine residue (C73S). Both the wild-type and mutant proteins were crystallized and their structures were determined at 100 K to a resolution of 1.62 A. The structure of the wild-type protein demonstrated that it was in the metaquo form, with the heme iron in the ferric oxidation state and the bound water lying 2.2 A from the heme iron. The structure of the C73S mutant protein was shown to contain a ferrous heme iron with a bound oxygen molecule. The bent bonding geometry of the Fe-O(1)-O(2) adduct results in a hydrogen bond of length 2.8 A between the second O atom, O(2), of molecular oxygen and N(2) of the distal histidine residue (His55) in both subunits contained within the asymmetric unit. This hydrogen-bonding interaction between His55 and the bound diatomic oxygen molecule provides new insight into the catalytic activation of H(2)O(2), which is essential for peroxidase activity.

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.

Pirin is a nuclear nonheme Fe protein of unknown function present in all human tissues. Here we describe that pirin may act as a redox sensor for the nuclear factor κB (NF-κB) transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to proinflammatory signals and controls the expression of a vast array of genes involved in immune and stress responses. Pirin's regulatory effect was tested with several metals and at different oxidations states, and our spectroscopic results show that only the ferric form of pirin substantially facilitates binding of NF-κB proteins to target κB genes, a finding that suggests that pirin performs a redox-sensing role in NF-κB regulation. The molecular mechanism of such a metal identity-and redox state-dependent regulation is revealed by our structural studies of pirin. The ferrous and ferric pirin proteins differ only by one electron, yet they have distinct conformations. The Fe center is shown to play an allosteric role on an R-shaped surface area that has two distinct conformations based on the identity and the formal redox state of the metal. We show that the R-shaped area composes the interface for pirin-NF-κB binding that is responsible for modulation of NF-κB's DNAbinding properties. The nonheme Fe protein pirin is proposed to serve as a reversible functional switch that enables NF-κB to respond to changes in the redox levels of the cell nucleus.metalloprotein | coregulator | reactive oxygen species (ROS) | oxidative stress | signal transduction activation

The enzyme dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a heme protein which has a globin fold but can function as both a hemoglobin and a peroxidase. As a peroxidase, DHP is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. As a hemoglobin, DHP cycles between the oxy and deoxy states as it reversibly binds oxygen for storage. Here, it is reported that the distal histidine, His55, exhibits conformational flexibility in the deoxy form and is consequently observed in two solvent-exposed conformations more than 9.5 A away from the heme. These conformations are analogous to the open conformation of sperm whale myoglobin. The heme iron in deoxy ferrous DHP is five-coordinate and has an out-of-plane displacement of 0.25 A from the heme plane. The observation of five-coordinate heme iron with His55 in a remote solvent-exposed conformation is consistent with the hypothesis that His55 interacts with heme iron ligands through hydrogen bonding in the closed conformation. Since His55 is also displaced by the binding of 4-iodophenol in an internal pocket, these results provide new insight into the correlation between heme iron ligation, molecular binding in the distal pocket and the conformation of the distal histidine in DHP.

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.