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A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative [Fe.sub.B] site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which Iowered the heme reduction potential by ~110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ~100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the [Fe.sub.B] site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues. biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering doi/10.1073/pnas.1000526107

This study demonstrates an improved magnetic protein microsphere-aided sandwich fluoroimmunoassay for the analysis of myoglobin and heart-type fatty acid binding protein (H-FABP), early protein markers associated with acute myocardial infarction. In preparation for the assay we constructed superparamagnetic human serum albumin (HSA)/[gamma]-[Fe.sub.2][O.sub.3] microspheres, and grafted capture antibodies (monoclonal antimyoglobin 7C3 and anti-H-FABP 10E1) onto the protein microspheres using the avidin--biotin system. Then the antibody-carrying microspheres were used in a sequential sandwich fluoroimmunoassay along with detection antibodies (Alexa fluor594-1abeled antimyoglobin 4E2 and FITC-labeled anti-H-FABP 9F3). The magnetic HSA/[gamma]-[Fe.sub.2][O.sub.3] microspheres were characterized by scanning electron microscopy, atomic force microscopy, Fourier transform infrared spectrophotometry, atomic absorption spectrophotometry, and vibrating sample magnetometry. Fluorescence images of the post-immunoassay microspheres recorded using an inverted fluorescence microscope showed that the average fluorescence intensity was correlated with the concentration of cardiac markers, in agreement with the results obtained by an F-4500 FL spectrophotometer; this indicated that the fluoroimmunoassay could be used to semiquantitatively detect both myoglobin and H-FABP. The detection limit was 10 ng/mL for myoglobin and 1 ng/mL for H-FABP.

pH-Dependent kinetic parameters ([k.sub.on], [k.sub.off], and [k.sub.cat]) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme--substrate (ES) complex ([k.sub.on]), the decay of the ES complex ([k.sub.off]), and the formation of the product ([k.sub.cat]) could be analyzed by transient kinetics as mass changes on the QCM plate. The [K.sub.d] ([k.sub.off]/[k.sub.on]) value was different from the Michaelis constant [K.sub.m] calculated from ([k.sub.off] + [k.sub.cat])/[k.sub.on], due to [k.sub.cat] > [k.sub.off]. The rate-determining step was the binding step ([k.sub.on]), and the catalytic rate ([k.sub.cat]) was faster than other [k.sub.on] and [k.sub.off] values. In the range of pH 2.5-5.0, values of [k.sub.on] gradually increased with decreasing pH showing a maximum at pH 3.7, values of [k.sub.off] were independent of pH, and [k.sub.cat] increased gradually with decreasing pH. As a result, the apparent rate constant ([k.sub.cat]/[K.sub.m]) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of [k.sub.on], is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of [k.sub.cat] at the lower pH correlated with the decrease of [alpha]-helix contents of the myoglobin substrate on the QCM.

In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the [y.sub.43.sup.+5] fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myogiobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility.

Sardines is the most important protein because it is responsible for the meat color is extracted from Pacific sardine and. Ethanol extraction proved to be more effective than ammonium sulfate extraction in the isolation of myoglobin (Mb) and for color analysis the results showed that Mb addition resulted in myosin denaturation and aggregation.

A study was carried out to investigate the effects of ionic strength and temperature on the interaction between fish myoglobin and myofibriliar proteins in a model system. Binding of myoglobin to natural actomyosin (NAM) was observed and it increased when ionic strength was increased at concentrations of 0.6 M KCL and with increases in temperature from 4 to 25 degree Celsius.

The synthesis and self-assembly behavior of biohybrid ABC triblock copolymers consisting of a synthetic diblock, polystyrene-b-polyethylene glycol, where m is varied, and a hemeprotein myoglobin (Mb) or horse radish peroxidase (HRP) are reported. It is revealed that the synthetic diblock copolymer is first functionalized with heme cofactor and then reconstituted with the apoprotein or the apoenzyme to give the protein-containing ABC triblock copolymer.

A molecular dynamics study is conducted to analyze the correlations between the nuclear volume, solvent-accessible surface and the folding state of various unfolded conformers of a [alpha] or a [beta] protein with any helical peptide. The results prove that the unfolded conformations need not be necessarily extended, as the volume variation does not affect the folding state to a large extent and hence is ignored in the study of the interiors of the protein.

Two mycocardial infarction biomarkers, myoglobin (MG) and cardiac troponin I (cTnI), were quantified at biological levels and in undiluted serum without sample pretreatment using surface plasmon resonance (SPR) sensors. To achieve detection of biomarkers in undiluted serum (72 mg/mL total protein concentration), minimization of the nonspecitic signal from the serum protein was achieved by immobilizing the antibody for the biomarkers on an N-hydroxysuccinimide activated 16-mercaptohexadecanoic acid self-assembled monolayer. This monolayer reduces the nonspecific signal from serum proteins in such a manner that short exposure of the sensor to serum prior to analysis prevents any further nonspecific adsorption during analysis. Thus, sensing of MG and cTnI was achieved on the basis of the difference between signals from the active sensor and a reference sensor that captured background interference. This resulted in direct measurement of these biomarkers in undiluted serum. Detection limits for both markers were below 1 ng/mL, which is below the threshold needed to detect myocardial infarction. Detecting biomarkers in the low ng/mL range without signal amplification in such a complex matrix as serum corresponds to a selectivity of [10.sup.8]. The root-mean-square-error (RMSE) of calibration was below 2 ng/mL.

The synthesis of three types of met- and zinc- myoglobin dyads, ZnMbAc(4)[Me.sup.+], ZnMbAc(6)[Me.sup.+], and metMbAc(6) is described by incorporating chemically modified metalloporphyrin cofactor appending an acridine(Ac) or a methylacridium ion ([[AcMe].sup.+]) into apo-Mb. It is shown that synthetic manipulation at the Mb surface with the use of an artificial DNA binder along with photoinduced reaction, can give insight to build new Mb-DNA complex and sensitive fluorescent for DNA.

A study shows that classical electrostatistics describes the quantitative description of, both the binding of the diatomic ligands XO (X= C. N, O) to the heme group in myoglobin and the dependence of their vibrational frequencies upon an external field by classical electrolysis. It is suggested that ligand binding occurs via an electrostatic bond, a generalization of the standard ionic bond to include induction, and that classical electrostatics can replace quantum mechanics for a considerable simplification of some complex problems.

A method for diffusion coefficient (D) measurement for proteins based on the pulsed laser-induced transient grating method using a photosensitive cross-linker is applied to characterize the pH denaturation process of holo- and apo-myoglobin (Mb) from the viewpoint of protein-water interaction. It is found that the pH denaturation curve monitored by D agrees quite well with that determined by the circular dichroism intensity for holo-Mb.

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous C[O.sub.2] IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.

The focus of the study is on requirements for a myoglobin (Mb) hemoglobin (Hb) functional model in aqueous solution, discrimination between [O.sub.2] and CO by hemoCDI, and the mechanism for autoxidation of oxy-hemoCDI. The results revealed the reason why hemoCDI acts as an excellent functional model for Mb and Hb that works in aqueous solution.

A demonstration of two-dimensional heterocorrelation analysis between spectrally resolved and temporally resolved fluorescene is presented to investigate the decay dynamics of the 8-anilino-1-naphthalenesulfonate-(ANS-) apomyoglobin complex. The relative decay dynamics across the emission spectra are resolved without measuring fluorescence lifetime at each wavelength as in the approach of decay-associated spectra.

Combining quantum and molecular mechanics (QM/MM) methods and protein structure prediction algorithms, helix and loop movements were computed along the pathway of CO dissociation from myoglobin (Mb). The principal effect of CO dissociation is shown to be a concerted rotation of the E and F halices, which hold the heme like a clamshell.

The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states is investigated by using experimental and computational methods. Fluorescence emission experiments and computational docking simulations have shown that the polarity probe NR binds to ApoMb within the heme binding pocket.

The applicability of Badger's rule to heme and non-heme iron-oxygen bonds is examined to gain insights into the protonation state of enzymatic ferryl species. The long Fe-O bonds reported in the crystal structures of the ferryl forms of myoglobin, cytochrome c peroxidase and catalase deviate substantially from the values predicted by Badger's rule, while the short Fe-O bonds obtained from X-ray absorption measurements are in good agreement with Badger's rule.

A previously undescribed spectrokinetic assay for the entry of water into the distal heine pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%, 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and [H.sub.2]O entry into the empty distal pocket of myoglobin are markedly different, 8 x [10.sup.7] and 2 x [10.sup.5] [M.sup.-1] x [s.sup.-1], respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry. distal water occupancy | spectrokinetic assay | Mb mutants | rebinding kinetics

Covalently linked films of the ferric heme protein myoglobin and poly-L-lysine graphite electrodes reacted with tert-butylhydroperoxide (tBuOOH) to form ferryloxy protein species according to Michaelis-Menten enzyme kinetics. Apparent kinetic constants are most likely governed by acidity-controlled protein conformations and their binding with tBuOOH in the intermediate protein-substrate complex.

The preparation of reconstituted myoglobin with the cobalt porphycene and O(sub 2) kinetics are presented. It is revealed that the constituted myoglobin with the cobalt porphycene has a high O(sub 2)affinity caused by its slow O(sub 2) dissociation.

The selectivity of electrochemical nitric oxide (NO) reduction by heme-didodecyldimethylammonium bromide (DDAB) and myoglobin-DDAB films as a function of potential and pH are examined. The selectivity of the pathway depends on whether a proton or NO molecule is transferred to a Fe(super II)-NO(super -) reaction intermediate and is influenced by pH, NO concentration and potential.

The photolysis and rebinding kinetics of carboxy-myoglobin (MbCO) embedded in a trehalose glass at room temperature using transient absorption and diffractive optics-based phase grating spectroscopy on nanosecond to microsecond time scale was investigated. The result suggests that protein structure plays a significant role in the bond energies at active sites which in turn provides a tuning element of the effective barrier heights independent to the transition state region.

CO rebinding kinetics after nanosecond photolysis of myoglobin encapsulated in wet silica gels exhibits an enhanced geminate phase that allows the determination of the microscopic rate constants and the activation barriers for distinct ligand docking sites inside the protein matrix. The germinate phase can be well-described by a biphasic lifetime distribution, using a maximum entropy method, reflecting rebinding from the distal and proximal sites.

The rebinding of CO to myoglobin (Mb) from locations around the active site is studied using a combination of molecular dynamics and stochastic simulations for native and L29F mutant Mb. It is observed that in the case of L29F mutant, the free-energy surface is flatter and the dynamics is much more rapid.

Ultrafast protein dynamics of the CO adduct of a myoglobin mutant with the polar distal histidine replaced by a nonpolar valine (H64V) are investigated by spectrally resolved infrared stimulated vibrational echo experiments and molecular dynamics (MD) simulations. It is observed that in aqueous solution at room temperature, the vibrational dephasing rate of CO in the mutant is reduced by approximately fifty percent relative to the native protein.

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long [C.sub.18]-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.

The dynamics of nitric oxide (NO) rebinding to Mb and its model system, microperoxidase, in D2O solution at 283 K after photolysis was directly observed by monitoring the stretching mode of NO using time-resolved IR spectroscopy is accurately determined. The transient spectra of photolyzed MbNO reveal two overlapping vibrational bands for bounds NO 1598 and 1611 cm(super -1).

Pulse radiolysis is used to study the kinetics and mechanism of the reaction of oxymyoblobin (MbFeO2) with NO2. The hemeproteins can be instrumental in detoxifying NO2 and hence in pre-empting deleterious processes, such as nitration of proteins.

9-287 GHz electron paramagnetic resonance (EPR) spectroscopy is used to examine the location of peroxyl radical in horse heart myoglobin (Mb), oxidized with H2O2. The high-field EPR spectra of the Mb peroxyl radical are simulated using a Hamiltonian, which describes the exchange and dipolar interaction between an oxoferryl iron and a protein radical.

Reaction of peroxynitrite with oxyMb and oxyHb in the presence of 1.2mM CO2 was investigated. Results reveal that this reaction can lead to Tyr, which might react with NO2 to generate 3-nitrotyrosine.

It is for the first time that orientational probability distribution function for CO transiently docked in myoglobin (Mb) and Hb is established. The ordered protein structure around the active site of the enzyme has a selectivity even for small ligands.

Mononuclear, dinuclear and tetranuclear artificial metalloproteases are prepared by attaching respective catalytic modules containing the Cu(II) complex of cyclen to a derivative of cross-linked polystyrene. The polymeric artificial metalloproteases effectively cleaved peptide bonds of myoglobin by hydrolysis.

Cross-linking of myoglobin (Mb) promoted by 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide within films of polystyrene sulfonate provided remarkable stabilization. Both the polyion film environment and cross-linking seem to play roles in stabilizing protein secondary structure and function at low pH.

The resonant Raman active mode identified in numerous studies as the heme iron-histidine stretch in the Raman spectrum of 15 exogenous ligands to the heme iron in the myoglobin proximal cavity mutant H93g is investigated. It is seen that Raman bands attributed to in-plane modes of the heme are unaffected by the change in heme-iron ligation.

Byline: Lone Bruun-Jensen (1), Laurette Sosniecki (1), L. H. Skibsted (1) Keywords: Key wordsaOxymyoglobin; Autoxidation; Pressure effects; Meat discoloration Abstract: aTwo opposing factors have been found to affect the rate of autoxidation of the fresh meat pigment oxymyoglobin, MbFe(II)O.sub.2, in air-saturated aqueous acetate buffer with increasing hydrostatic pressure to yield metmyoglobin, MbFe(III): (1) expansion in the transition state, corresponding to a volume of activation of IV = +12.7+-0.6 ml mol.sup.--1 (25.0degC, 0.16 M NaCl), in effect decreasing the rate and (2) an increased specific acid catalysis resulting from a decrease in solution pH and increasing the rate. In the pressure range investigated (up to 250 MPa) and at the pH relevant to traditionally deboned meat (pH = 5.65 at ambient pressure investigated, together with pH = 5.21 in order to obtain mechanistic information), the pressure/pH effect was found almost to negate the kinetic effect of expansion in the transition state. Red-to-brown discoloration during pressure treatment of meat is accordingly expected to be less significant for hot-deboned and other high-pH meats. Author Affiliation: (1) Department of Dairy and Food Science, Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark, DK Article note: Received: 7 April 1997 / Revised version: 2 June 1997

The genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in Escherichia coli is described by evolving an orthogonal tTNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical ' handle' for fluorescent labeling of p-Tpa-encoded myoglobin by the photoclick reaction. The structural basis for the biosynthetic incorporation of p-Tpa into proteins is analyzed by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa.

A conserved 2-His-1-Glu metal center, which is found in natural nonheme iron-containing enzymes, is engineered into sperm whale myoglobin by replacing by replacing Leu29 and Phe43 with Glu and His. The analysis has presented a novel protein model of NOR and has provided insights into a newly discovered member of the NOR family, gNOR.

GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants are measured to understand the discrepancies during the unfolding of wild-type holomyoglobin in the ferric state (metMb). The high affinities of native apoMb (N) for hemin are found to have significant influence on the stability of holo-metMb and the partially unfolded intermediate with hemin bound ((IH) exhibiting a hemichrome spectra are indicative of a bis-histidyl axial coordination which is seen clearly when the stability of the N state or its affinity for hemin is reduced.

Correlated single-crystal spectroscopy with crystallography was employed to characterize the single crystal of the nitrite adduct of ferric myoglobin (Mb) exposed to high-intensity synchrotron X-ray radiation. The 1.55 [Angstrom] resolution crystal structure of the photoproduct reveals retention of the O-binding mode for binding of nitrite to the iron center.

A pair of myoglobins containing inherently distorted [alpha]-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared to study the functional consequence of intrinsic heme deformation. A distinctive controlling mechanism observed for ligand binding by the deformed heme revealed that the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

Backbone-modified phosphorothioate DNA and biotin- and maleimide-containing bifunctional linkers are used to achieve controlled alignment of streptavidin (STV), myoglobin, and nanoparticles with nanometer resolution. The proposed method which facilitates the high yield, nanoscale-precision conjugation of multiple proteins to DNA could be used to design a linker to conjugate any proteins and nanomaterials specifically for a wide range of applications.

The pathways identified by alkyl isocyanides (CNRs) for diatomic ligand movement into and out of Mb, with their side chains acting as transition state analogues are reviewed. The strong correlation observed between the fraction of in conformer, [F.sub.in], for long-chain MbCNR complexes, the fraction of geminate recombination of photodissociated [O.sub.2], and the bimolecular rates of [O.sub.2] entry into the distal pocket revealed that alkyl isocyanides serve as transition state analogues for the movement of [O.sub.2] into and out of the binding pocket of Mb.

A study is conducted to gain insight into the low- and high-frequency peaks observed for the alkyl isocyanides upon binding to sperm whale myoglobin (Mb). The internal rebinding for in alkyl side chain conformation is promoted by the photodissociated ligand which is stuck in the distal pocket while the out conformation that inhibited geminate recombination is attributed to the part of the ligand that is already in an open E7 channel, poised for rapid escape.