Detection of Ras GTPase protein radicals through immuno-spin trapping.

Over the past decade immuno-spin trapping (IST) has been used to detect and identify protein radical sites in numerous heme and metalloproteins. To date, however, the technique has had little application toward nonmetalloproteins. In this study, we demonstrate the successful application of IST in a system free of transition metals and present the first conclusive evidence of (•)NO-mediated protein radical formation in the HRas GTPase. HRas is a nonmetalloprotein that plays a critical role in regulating cell-growth control. Protein radical formation in Ras GTPases has long been suspected of initiating premature release of bound guanine nucleotide. This action results in altered Ras activity both in vitro and in vivo. As described herein, successful application of IST may provide a means for detecting and identifying radical-mediated Ras activation in many different cancers and disease states in which Ras GTPases play an important role.

Regulation of Ras proteins by reactive nitrogen species.

Ras GTPases have been a subject of intense investigation since the early 1980s, when single point mutations in Ras were shown to cause deregulated cell growth control. Subsequently, Ras was identified as the most prevalent oncogene found in human cancer. Ras proteins regulate a host of pathways involved in cell growth, differentiation, and apoptosis by cycling between inactive GDP-bound and active GTP-bound states. Regulation of Ras activity is controlled by cellular factors that alter guanine nucleotide cycling. Oncogenic mutations prevent protein regulatory factors from down-regulating Ras activity, thereby maintaining Ras in a chronically activated state. The central dogma in the field is that protein modulatory factors are the primary regulators of Ras activity. Since the mid-1990s, however, evidence has accumulated that small molecule reactive nitrogen species (RNS) can also influence Ras guanine nucleotide cycling. Herein, we review the basic chemistry behind RNS formation and discuss the mechanism through which various RNS enhance nucleotide exchange in Ras proteins. In addition, we present studies that demonstrate the physiological relevance of RNS-mediated Ras activation within the context of immune system function, brain function, and cancer development. We also highlight future directions and experimental methods that may enhance our ability to detect RNS-mediated activation in cell cultures and in vivo. The development of such methods may ultimately pave new directions for detecting and elucidating how Ras proteins are regulated by redox species, as well as for targeting redox-activated Ras in cancer and other disease states.

Dehaloperoxidase-hemoglobin from Amphitrite ornata is primarily a monomer in solution.

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.

Internal binding of halogenated phenols in dehaloperoxidase-hemoglobin inhibits peroxidase function.

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.

Determination of separate inhibitor and substrate binding sites in the dehaloperoxidase-hemoglobin from Amphitrite ornata.

Dehaloperoxidase-hemoglobin (DHP A) is a dual function protein found in the terrebellid polychaete Amphitrite ornata. A. ornata is an annelid, which inhabits estuary mudflats with other polychaetes that secrete a range of toxic brominated phenols. DHP A is capable of binding and oxidatively dehalogenating some of these compounds. DHP A possesses the ability to bind halophenols in a distinct, internal distal binding pocket. Since its discovery, the distal binding pocket has been reported as the sole binding location for halophenols; however, data herein suggest a distinction between inhibitor (monohalogenated phenol) and substrate (trihalogenated phenol) binding locations. Backbone (13)Calpha, (13)Cbeta, carbonyl (13)C, amide (1)H, and amide (15)N resonance assignments have been made, and various halophenols were titrated into the protein. (1)H-(15)N HSQC experiments were collected at stoichiometric intervals during each titration, and binding locations specific for mono- and trihalogenated phenols have been identified. Titration of monohalogenated phenol induced primary changes around the distal binding pocket, while introduction of trihalogenated phenols created alterations of the distal histidine and the local area surrounding W120, a structural region that corresponds to a possible dimer interface region recently observed in X-ray crystal structures of DHP A.

Different modes of binding of mono-, di-, and trihalogenated phenols to the hemoglobin dehaloperoxidase from Amphitrite ornata.

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.

Determinants of substrate internalization in the distal pocket of dehaloperoxidase hemoglobin of Amphitrite ornata.

Dehaloperoxidase (DHP) is a small heme protein in the coelom of the terebellid polychaete Amphitrite ornata. It can act both as an oxygen storage protein (hemoglobin function) and as a dehaloperoxidase (peroxidase function). The X-ray structure of the ferric form shows that the phenolic substrate can bind inside the protein, which is not the case for a typical peroxidase. In the present study, we have used CO-ligated DHP to mimic the distal pocket of the peroxidase DHP and to probe under which conditions both a halophenol and a diatomic ligand can be accommodated in the distal pocket. To vary the structure of the distal pocket, we have compared wild-type DHP and mutants H55V and H55R at different pH values, using flash photolysis in the visible and FTIR spectroscopy in the CO stretching bands. The latter technique is extremely sensitive to even small structural changes in the CO environment and thus can report substrate binding in the distal pocket. Our results on wild-type DHP and its variants indicate that halophenols and a diatomic ligand can indeed simultaneously be present in the distal pocket if the distal histidine is in the low-pH conformation, in which its side chain is swung out of the distal pocket. The markedly different pH dependencies of enzyme activity and substrate binding are not consistent with the hypothesis that substrate dehalogenation occurs within the interior of DHP.

Conformational dynamics associated with photodissociation of CO from dehaloperoxidase studied using photoacoustic calorimetry.

Herein, we present photoacoustic calorimetry and transient absorption studies of the dynamics and energetics associated with dissociation of a ligand from Fe(2+) dehaloperoxidase (DHP) from Amphitrite ornata. Our data show that CO photodissociation is associated with an endothermic (DeltaH = 8 +/- 3 kcal mol(-1)) volume expansion (DeltaV = 9.4 +/- 0.6 mL mol(-1)) that occurs within 50 ns upon photodissociation. No additional thermodynamics were detected on slower time scales (up to 10 micros), suggesting that the dissociated ligand rapidly escapes from the heme-binding pocket into the surrounding solvent. Similar volume and enthalpy changes were observed for CO photodissociation in the presence of the substrate, 2,4-dichlorophenol or 4-bromophenol, indicating that either the substrate does not bind in the protein distal cavity at ambient temperature or its presence does not impact the thermodynamic profile associated with ligand dissociation. We attribute a fast ligand exchange between the protein active site and the surrounding solvent to the high flexibility of the distal histidine residue, His55, that provides a direct pathway between the heme-binding pocket and the protein exterior. The dynamics and energetics of conformational changes observed for dissociation of a ligand from DHP differ significantly from those measured previously for photodissociation of CO from the structural homologue myoglobin, suggesting that structural dynamics in DHP are fine-tuned to enhance the peroxidase function of this protein.

X-ray crystal structural analysis of the binding site in the ferric and oxyferrous forms of the recombinant heme dehaloperoxidase cloned from Amphitrite ornata.

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.

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase.

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.

Enzyme function of the globin dehaloperoxidase from Amphitrite ornata is activated by substrate binding.

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.