Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

Low-frequency EPR of ferrimyoglobin fluoride and ferrimyoglobin cyanide: a case study on the applicability of broadband analysis to high-spin hemoproteins and to HALS hemoproteins.

An EPR spectrometer has been developed that can be tuned to many frequencies in the range of ca 0.1-15 GHz. Applicability has been tested on ferrimyoglobin fluoride (MbF) and ferrimyoglobin cyanide (MbCN). MbF has a high-spin (S = 5/2) spectrum with 19F superhyperfine splitting that is only resolved in X-band along the heme normal. Low-frequency EPR also resolves the splitting in the heme plane. Measurement of linewidth as a function of frequency provides the basis for an analysis of inhomogeneous broadening in terms of g-strain, zero-field distribution, unresolved superhyperfine splittings and dipolar interaction. Rhombicity in the g tensor is found to be absent. MbCN (S = 1/2) has a highly anisotropic low spin (HALS) spectrum for which gx cannot be determined unequivocally in X-band. Low-frequency EPR allows for measurement of the complete spectrum and determination of the g-tensor.

Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin.

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb.

X-ray-induced photoreduction of heme metal centers rapidly induces active-site perturbations in a protein-independent manner.

Since the advent of protein crystallography, atomic-level macromolecular structures have provided a basis to understand biological function. Enzymologists use detailed structural insights on ligand coordination, interatomic distances, and positioning of catalytic amino acids to rationalize the underlying electronic reaction mechanisms. Often the proteins in question catalyze redox reactions using metal cofactors that are explicitly intertwined with their function. In these cases, the exact nature of the coordination sphere and the oxidation state of the metal is of utmost importance. Unfortunately, the redox-active nature of metal cofactors makes them especially susceptible to photoreduction, meaning that information obtained by photoreducing X-ray sources about the environment of the cofactor is the least trustworthy part of the structure. In this work we directly compare the kinetics of photoreduction of six different heme protein crystal species by X-ray radiation. We show that a dose of ∼40 kilograys already yields 50% ferrous iron in a heme protein crystal. We also demonstrate that the kinetics of photoreduction are completely independent from variables unique to the different samples tested. The photoreduction-induced structural rearrangements around the metal cofactors have to be considered when biochemical data of ferric proteins are rationalized by constraints derived from crystal structures of reduced enzymes.

Antioxidant, Antimicrobial and Metmyoglobin Reducing Activity of Artichoke (Cynara scolymus) Powder Extract-Added Minced Meat during Frozen Storage.

The present study aimed to investigate the bioactive compounds in artichoke (Cynara scolymus) powder, having antioxidant and antimicrobial activity, and to determine the effectiveness of artichoke (C. scolymus) powder extract within the minced meat. C. scolymus was extracted using two different methods. The method incorporating high phenolic and flavonoid content levels was used in other analyses and the phenolic and flavonoid contents in C. scolymus extract was determined using LC-QTOF-MS. Antioxidant, antimicrobial, and metmyoglobin (metMb) reducing activities and pH values of the extract-added minced meat samples were measured for 10 days during storage. DPPH, FRAP, and ABTS were used in the antioxidant analyses. The antimicrobial activity of C. scolymus extract was evaluated on five different food pathogens by using the disc diffusion method. The most resistant bacterium was found to be Listeria monocytogenes (18.05 mm ± 0.24). The amount of metMb was measured in the minced meat sample that was added to the extract during storage (p < 0.05). MetMb formation and pH value on the sixth day of storage were found to be at lower levels than in the control group. In conclusion, C. scolymus exhibited a good antimicrobial and antioxidant effect and can be used in storing and packaging the food products, especially the meat and meat products.

In Silico Insight into the Reductive Nitrosylation of Ferric Hemeproteins.

A combination of in silico methods was used to extend the experimental description of the reductive nitrosylation mechanism in ferric hemeproteins with the molecular details of the role of surrounding amino acids. The computational strategy consisted in the estimation of potential energy profiles for the transition process associated with the interactions of the coordinated N(NO) moiety with O(H2O) or O(OH-) as nucleophiles, and with distal amino acids as proton acceptors or affecting the stability of transition states. We inspected the reductive nitrosylation in three representative hemeproteins -sperm whale metmyoglobin, α subunit of human methemoglobin and nitrophorin 4 of Rhodnius prolixus. For each case, classical molecular dynamics simulations were performed in order to obtain relevant reactive conformations, and a potential energy profile for the reactive step was obtained using adiabatic mapping or nudged elastic band approaches at the QM/MM level. Specifically, we report the role of a charged Arg45 of myoglobin in destabilizing the transition state when H2O acts as nucleophile, differently to the neutral Pro43 of the hemoglobin subunit. The case of the nitrophorin is unique in that the access of the required water molecules is scarce, thus, preventing the reaction.

Interrelationship between myoglobin oxidation and lipid oxidation during the processing of Cantonese sausage with d-sodium erythorbate.

BACKGROUND: Pork is used as raw material to produce Cantonese sausage, and 0.5 or 1 g kg-1 of d-sodium erythorbate is added to the pork meat. In this study the myoglobin oxidation rate, relative metmyoglobin content, heme iron content, redness, pH, free radical content and thiobarbituric acid reactive substance (TBARS) value were measured at different processing times and different content of d-sodium erythorbate. RESULTS: It was found that d-sodium erythorbate significantly reduced the free radical content and myoglobin and lipid oxidation rates and increased heme iron levels. When d-sodium erythorbate was added to the sausage, the absorption peak of myoglobin porphyrin shifted left, migrating from 414 to 405 nm. At 72 h, with an increase in the d-sodium erythorbate content, a significant negative correlation was identified between heme iron and the degree of redness (P < 0.01). CONCLUSION: During sausage processing, there are strong correlations among TBARS values, free radical content, metmyoglobin levels, heme iron levels, a* and pH at the same d-sodium erythorbate level. At the same processing time, adding d-sodium erythorbate can slow the rate of myoglobin and lipid oxidation and prevent the discoloration of sausage. © 2019 Society of Chemical Industry.

Effect of high pressure treatment on the color of fresh and processed meats: A review.

High pressure (HP) treatment often results in discoloration of beef, lamb, pork, and poultry. The degree of color changes depends on the physical and chemical state of the meat, especially myoglobin, and the atmospheric conditions during and after pressurization. A decreased redness is attributed to a large degree to the oxidation of the bright red oxymyoglobin or the purplish deoxymyoglobin into the brownish metmyoglobin, as well as to the denaturation of myoglobin. Surely, the high myoglobin content makes beef more exposed to this discoloration compared to the white chicken meat. In addition, HP treatment causes denaturation of myofibrillar proteins followed by aggregation, consequently, changing the surface reflectance and increasing lightness. Other intrinsic and extrinsic factors may affect the pressure-induced color changes positively or negatively. In this review, the pressure-induced color changes in meat are discussed in relation to modification of the myoglobin molecule, changes in the meat microstructure, and the impact of the presence of different chemical compounds and physical conditions during processing.

Comparative study of the influence of hawthorn (Crataegus monogyna) berry ethanolic extract and butylated hydroxylanisole (BHA) on lipid peroxidation, myoglobin oxidation, consistency and firmness of minced pork during refrigeration.

BACKGROUND: Following public concern on the use of synthetic food antioxidants, there is an increasing demand for the application of mixed or purified natural antioxidants to maintain quality of meat products quality during storage. The aim of this research was to investigate the effect of ethanolic extract of hawthorn berry, compared to butylated hydroxylanisole (BHA), on lipid peroxidation, myoglobin oxidation, protein electrophoresis pattern, consistency and firmness of minced pork during refrigeration at 4 °C, and to identify the relationship between chemical modifications and consistency variation. RESULTS: After 6 days of refrigeration it was found that the thiobarbituric acid reactive substances value of minced pork containing 200 mg GAE kg-1 total phenolics in minced meat (200 HP) was significantly lower (0.1543 ± 0.006 mg) compared to BHA-treated meat. The ratio of oxymyoglobin to metmyoglobin in treated minced pork was respectively 0.845 for 200 HP and 0.473 for BHA-treated minced meat. Concentrations of 100 HP or 300 HP will generate statistically higher firmness than BHA in minced pork. CONCLUSION: Hawthorn berry ethanolic extract was more effective than BHA in reducing lipid oxidation and protein degradation, for maintaining firmness and consistency of minced pork during 6 days of refrigeration at 4 °C. © 2017 Society of Chemical Industry.

Mechanisms of HNO Reactions with Ferric Heme Proteins.

Many HNO-scavenging pathways exist to regulate its biological and pharmacological activities. Such reactions often involve ferric heme proteins and form an important basis for HNO probe development. However, mechanisms of HNO reactions with ferric heme proteins are largely unknown. We performed a computational investigation using metmyoglobin and catalase as representative ferric heme proteins with neutral and negatively charged axial ligands to provide the first detailed pathways. The results reproduced experimental barriers well with an average error of 0.11 kcal mol-1 . The rate-limiting step was found to be dissociation of the resting ligand or HNO coordination when there is no resting ligand. For both heme proteins, in contrast to the non-heme case, the reductive nitrosylation step was found to be barrierless proton-coupled electron transfer, which provides the major thermodynamic driving force for the overall reaction. The origin of the difference in reactivity between metmyoglobin and catalase was also revealed.

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

BACKGROUND: Metmyoglobin (MbFe(III)) reaction with H(2)O(2) has been a subject of study over many years. H(2)O(2) alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H(2)O(2) dismutation associated with MbFe(III) inactivation remains obscure. METHODS: MbFe(III) reaction with excess H(2)O(2) in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H(2)O(2) depletion, O(2) evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. RESULTS: The rates of both H(2)O(2) dismutation and heme inactivation processes depend on [MbFe(III)], [H(2)O(2)] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H(2)O(2) molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H(2)O(2) and by nitroxide, respectively. CONCLUSIONS: The nitroxide effects on H(2)O(2) dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H(2)O(2) is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H(2)O(2) adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1. GENERAL SIGNIFICANCE: The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.

Genome-wide expression profiling in muscle and subcutaneous fat of lambs in response to the intake of concentrate supplemented with vitamin E.

BACKGROUND: The objective of this study was to acquire a broader, more comprehensive picture of the transcriptional changes in the L. Thoracis muscle (LT) and subcutaneous fat (SF) of lambs supplemented with vitamin E. Furthermore, we aimed to identify novel genes involved in the metabolism of vitamin E that might also be involved in meat quality. In the first treatment, seven lambs were fed a basal concentrate from weaning to slaughter (CON). In the second treatment, seven lambs received basal concentrate from weaning to 4.71 ± 2.62 days and thereafter concentrate supplemented with 500 mg dl-α-tocopheryl acetate/kg (VE) during the last 33.28 ± 1.07 days before slaughter. RESULTS: The addition of vitamin E to the diet increased the α-tocopherol muscle content and drastically diminished the lipid oxidation of meat. Gene expression profiles for treatments VE and CON were clearly separated from each other in the LT and SF. Vitamin E supplementation had a dramatic effect on subcutaneous fat gene expression, showing general up-regulation of significant genes, compared to CON treatment. In LT, vitamin E supplementation caused down-regulation of genes related to intracellular signaling cascade. Functional analysis of SF showed that vitamin E supplementation caused up-regulation of the lipid biosynthesis process, cholesterol, and sterol and steroid biosynthesis, and it down-regulated genes related to the stress response. CONCLUSIONS: Different gene expression patterns were found between the SF and LT, suggesting tissue specific responses to vitamin E supplementation. Our study enabled us to identify novel genes and metabolic pathways related to vitamin E metabolism that might be implicated in meat quality. Further exploration of these genes and vitamin E could lead to a better understanding of how vitamin E affects the oxidative process that occurs in manufactured meat products.

A wave-mechanical model of incoherent quasielastic scattering in complex systems.

Quasielastic incoherent neutron scattering (QENS) is an important tool for the exploration of the dynamics of complex systems such as biomolecules, liquids, and glasses. The dynamics is reflected in the energy spectra of the scattered neutrons. Conventionally these spectra are decomposed into a narrow elastic line and a broad quasielastic band. The band is interpreted as being caused by Doppler broadening due to spatial motion of the target molecules. We propose a quantum-mechanical model in which there is no separate elastic line. The quasielastic band is composed of sharp lines with twice the natural line width, shifted from the center by a random walk of the protein in the free-energy landscape of the target molecule. The walk is driven by vibrations and by external fluctuations. We first explore the model with the Mössbauer effect. In the subsequent application to QENS we treat the incoming neutron as a de Broglie wave packet. While the wave packet passes the protons in the protein and the hydration shell it exchanges energy with the protein during the passage time of about 100 ns. The energy exchange broadens the ensemble spectrum. Because the exchange involves the free-energy landscape of the protein, the QENS not only provides insight into the protein dynamics, but it may also illuminate the free-energy landscape of the protein-solvent system.

Comment on "Sensitive marker bands for the detection of spin states of heme in surface-enhanced resonance Raman scattering spectra of metmyoglobin" by Y. Kitahama, M. Egashira, T. Suzuki, I. Tanabe and Y. Ozaki.

We contrast recently reported surface-enhanced resonance Raman spectra (SERRS) of myoglobin on silver nanoparticles with established knowledge about this complex. We conclude that the detected bands are not related to the spin states of the protein cofactor, being rather originated by a heme coordination change induced by the metal surface.

Reply to the comment on "Sensitive marker bands for the detection of spin states of heme in surface-enhanced resonance Raman scattering spectra of metmyoglobin".

In our SERRS spectra of metmyoglobin by excitation at 514 nm, the peak at 1510 cm(-1), which is assigned to the 6-coordinated heme in the low spin state, was observed by the addition of imidazole and NaN3. Thus, the SERRS likely originates not from the non-native 5-coordinated heme, which is in the high spin state.

Caesalpinia decapetala Extracts as Inhibitors of Lipid Oxidation in Beef Patties.

In this study we investigated the effects of Caesalpinia decapetala (CD) extracts on lipid oxidation in ground beef patties. Plant extracts and butylated hydroxytoluene (BHT) were individually added to patties at both 0.1% and 0.5% (w/w) concentrations. We assessed the antioxidant efficacy of CD by the ferric reducing antioxidant power (FRAP) assay and evaluated their potential as natural antioxidants for meat preservation by thiobarbituric acid reactive substance (TBARS) values, hexanal content, fatty acid composition and color parameters. These were tested periodically during 11 days of refrigerated storage. TBARS levels were significantly lower (p ≤ 0.05) in the samples containing plant extracts or BHT than in the non-treated control. In addition, the beef patties formulated with the selected plant extracts showed significantly (p ≤ 0.05) better color stability than those without antioxidants. These results indicate that edible plant extracts are promising sources of natural antioxidants and can potentially be used as functional preservatives in meat products.

The relationship between muscle α-tocopherol concentration and meat oxidation in light lambs fed vitamin E supplements prior to slaughter.

BACKGROUND: The use of concentrates supplemented with α-tocopherol in animals is an effective method to reduce the oxidative processes that occur in meat products. The high cost of α-tocopherol requires accurate feeding, so it is necessary to define the minimum period of α-tocopherol concentrate supplementation that will ensure an acceptable meat quality. Indoor concentrate-fed light lambs (n = 35) were supplemented with 500 mg dl-α-tocopheryl acetate (VE) kg(-1) concentrate for a period of between 4 and 28 days before being slaughtered at 22-24 kg body weight. Control lambs (n = 12) were not supplemented with α-tocopherol. RESULTS: The α-tocopherol content in both plasma and muscle tissues increased significantly with the length of supplementation (P < 0.001). The thiobarbituric acid-reactive substance (TBARS) concentration in meat decreased exponentially when the muscle α-tocopherol concentration was increased to 0.61-0.90 mg kg(-1) fresh meat (P < 0.05). After 7 days of display, the formation of metmyoglobin (MMb) decreased significantly as the α-tocopherol content increased to 0.31-0.60 mg kg(-1) meat (P < 0.05). CONCLUSION: A range of 0.61-0.90 mg α-tocopherol kg(-1) fresh meat protected fresh lamb meat from lipid oxidation and MMb formation. This level can be achieved by supplementation with 500 mg VE kg(-1) concentrate for a period of 7-14 days before slaughter.

[Spectral Study on Coordination Reaction Between metMyoglobin and Nitric Oxide].

As we all known, the instantaneous reaction between protein and ligands are very important to adjust the normal playing of biological function. And nitric oxide interactions with iron are the most important biological reactions in which NO participates. Unlike carbon monoxide or oxygen, NO can also bind reversibly to ferric iron. In this paper, UV-Vis absorption and CD spectra were used to study coordination reaction process between horse heart metMb and NO, to demonstrate the coordination reaction mechanism and to explore the influencing factors of metMb with NO. The experimental results showed that metMb could react with NO, and obtained three new peaks at 420 nm, 534 and 568 nm, respectively, which implied metMb and NO have reacted and generated a new complex-nitrosylmetmyoglobin (metMbNO). Then as time went on, NO concentration decreased in the solution, and the Fe-N bond fractured under the attack of H2O, then NO leaves slowly from metMbNO, and met-Mb was regenerated. In this experiment, we also found that external conditions such as buffer medium, ionic strength, pH, temperature, etc, had an important influence on the coordination reaction between metMb and NO. It was favorable for the coordination reaction, when the 0.01 mol x L(-1) phosphate buffer. solution is near neutral condition, the temperature is 280 K, the coordination reaction could reach equilibrium at a fastest speed. In addition, the CD date show that NO only reacts with Fe atom in the center of heme and has less effect on the secondary structuers of protein. The research of metMb and NO played an important role to further study the function of NO. Especially the establish of equilibrium reaction mechanism between NO and heme protein has an important research value on maintaining the balance of NO in vivo and keeping the normal function in the body's cells.

Protein electronic conductors: hemin-substrate bonding dictates transport mechanism and efficiency across myoglobin.

Electron transport (ETp) across met-myoglobin (m-Mb), as measured in a solid-state-like configuration between two electronic contacts, increases by up to 20 fold if Mb is covalently bound to one of the contacts, a Si electrode, in an oriented manner by its hemin (ferric) group, rather than in a non-oriented manner. Oriented binding of Mb is achieved by covalently binding hemin molecules to form a monolayer on the Si electrode, followed by reconstitution with apo-Mb. We found that the ETp temperature dependence (>120 K) of non-oriented m-Mb virtually disappears when bound in an oriented manner by the hemin group. Our results highlight that combining direct chemical coupling of the protein to one of the electrodes with uniform protein orientation strongly improves the efficiency of ET across the protein. We hypothesize that the behavior of reconstituted m-Mb is due to both strong protein-substrate electronic coupling (which is likely greater than in non-oriented m-Mb) and direct access to a highly efficient transport path provided by the hemin group in this configuration.

Self crowding of globular proteins studied by small-angle x-ray scattering.

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D2O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10(-4) cm(3)mol/g(2) for Mb in phosphate buffer, 1.6 ×10(-4) cm(3)mol/g(2) for BPTI in phosphate buffer and 9.2 ×10(-4) cm(3)mol/g(2) for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.