Probing protein adsorption onto mercaptoundecanoic acid stabilized gold nanoparticles and surfaces by quartz crystal microbalance and zeta-potential measurements.

The adsorption characteristics of three proteins [bovine serum albumin (BSA), myoglobin (Mb), and cytochrome c (CytC)] onto self-assembled monolayers of mercaptoundecanoic acid (MUA) on both gold nanoparticles (AuNP) and gold surfaces (Au) are described. The combination of quartz crystal microbalance measurements with dissipation (QCM-D) and pH titrations of the zeta-potential provide information on layer structure, surface coverage, and potential. All three proteins formed adsorption layers consisting of an irreversibly adsorbed fraction and a reversibly adsorbed fraction. BSA showed the highest affinity for the MUA/Au, forming an irreversibly adsorbed rigid monolayer with a side-down orientation and packing close to that expected in the jamming limit. In addition, BSA showed a large change in the adsorbed mass due to reversibly bound protein. The data indicate that the irreversibly adsorbed fraction of CytC is a monolayer structure, whereas the irreversibly adsorbed Mb is present in form of a bilayer. The observation of stable BSA complexes on MUA/AuNPs at the isoelectric point by zeta-potential measurements demonstrates that BSA can sterically stabilize MUA/AuNP. On the other hand, MUA/AuNP coated with either Mb or CytC formed a reversible flocculated state at the isoelectric point. The colloidal stability differences may be correlated with weaker binding in the reversibly bound overlayer in the case of Mb and CytC as compared to BSA.

The pH dependence of the activity of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first hemoglobin that has peroxidase activity as part of its native function. The substrate 2,4,6-tribromophenol (TBP) is oxidatively debrominated by DHP to form 2,6-dibromoquinone (DBQ) in a two-electron process. There is a well-defined internal binding site for TBP above the heme, a feature not observed in other hemoglobins or peroxidases. A study of the pH dependence of the activity of DHP reveals a substantial difference in mechanism. From direct observation of the Soret band of the heme it is shown that the pKa for heme activation in protein DHP is 6.5. Below this pH the heme absorbance decreases in the presence of H2O2 with or without addition of substrate. The low pH data are consistent with significant heme degradation. Above pH 6.5 addition of H2O2 causes the heme to shift rapidly to a compound II spectrum and then slowly to an unidentified intermediate with an absorbance of 410 nm. However, the pKa of the substrate TBP is 6.8 and the greatest enzyme activity is observed above the pKa of TBP under conditions where the substrate is a phenolate anion (TPBO-). Although the mechanisms may differ, the data show that both neutral TBP and anionic TPBO- are converted to the quinone product. The mechanistic implications of the pH dependence are discussed by comparison other known peroxidases, which oxidize substrates at the heme edge.

Resonance Raman study of ferric heme adducts of dehaloperoxidase from Amphitrite ornata.

The study of axial ligation by anionic ligands to ferric heme iron by resonance Raman spectroscopy provides a basis for comparison of the intrinsic electron donor ability of the proximal histidine in horse heart myoglobin (HHMb), dehaloperoxidase (DHP), and horseradish peroxidase (HRP). DHP is a dimeric hemoglobin (Hb) originally isolated from the terebellid polychaete Amphitrite ornata. The monomers are structurally related to Mb and yet DHP has a peroxidase function. The core size marker modes, v2 and v3, were observed using Soret excitation, and DHP-X was compared to HHMb-X for the ligand series X = F, Cl, Br, SCN, OH, N3, and CN. Special attention was paid to the hydroxide adduct, which is also formed during the catalytic cycle of peroxidases. The Fe-OH stretching frequency was observed and confirmed by deuteration and is higher in DHP than in HHMb. The population of high-spin states of the heme iron in DHP was determined to be intermediate between HHMb and HRP. The data provide the first direct measurement of the effect of axial ligation on the heme iron in DHP. The Raman data support a modified charge relay in DHP, in which a strongly hydrogen-bonded backbone carbonyl (>C=O) polarizes the proximal histidine. The charge relay mechanism by backbone carbonyl >C=O-His-Fe is the analogue of the Asp-His-Fe of peroxidases and Glu-His-Fe of flavohemoglobins.

Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin.

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.

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.

Spectroscopic study of substrate binding to the carbonmonoxy form of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) is a globular heme enzyme found in the marine worm Amphitrite ornata that can catalyze the dehalogenation of halophenols to the corresponding quinones by using hydrogen peroxide as a cosubstrate. Its three-dimensional fold is surprisingly similar to that of the oxygen storage protein myoglobin (Mb). A key structural feature common to both DHP and Mb is the existence of multiple conformations of the distal histidine. In DHP, the conformational flexibility may be involved in promotion of substrate and cosubstrate entry and exit. Here we have explored the dynamics of substrate binding in DHP using Fourier transform infrared spectroscopy and flash photolysis. A number of discrete conformations at the active site were identified from the appearance of multiple CO absorbance bands in the infrared region of the spectrum. Upon photolysis at cryogenic temperatures, the CO molecules are trapped at docking sites within the protein matrix, as inferred from the appearance of several photoproduct bands characteristic of each site. Substrate binding stabilizes the protein by approximately 20 kJ/mol. The low yield of substrate-bound DHP at ambient temperature points toward a steric inhibition of substrate binding by carbon monoxide.

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.

Covalent attachment of a nickel nitrilotriacetic acid group to a germanium attenuated total reflectance element.

The surface of a germanium internal reflectance element (IRE) was modified to bind 6X-histidine (his)-tagged biomolecules. The step-by-step surface modification was monitored via single-pass attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR). Initially an adlayer of 7-octenyltrimethoxysilane (7-OTMS) was formed on the Ge crystal through the surface hydroxyl groups, which were produced via ozonolysis of the Ge surface. The vinyl moiety of 7-OTMS was oxidized to a carboxylic acid, which was activated by 1,1'-carbonydiimidazole (CDI) to produce a labile imidazole. The labile imidazole that resulted from the CDI coupling was then displaced by the primary amine of nitrilotriacetic acid (NTA). Nickel sulfate was added to the system, and it coordinated with the three carbonyl groups and the nitrogen on NTA, thus leaving the ability of Ni to coordinate with two adjacent histidine residues. Binding of his-tagged biotin to nickel nitrilotriacetic acid (Ni-NTA) was observed by ATR-FT-IR spectroscopy. The surface modification method presented in this paper had minimal nonspecific binding, the Ni-NTA surface was reusable if stored properly, and complete removal of the organic surface was achievable.