Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats

The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF_2α), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of ω-6- vs. ω-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF_2α was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk.Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.     

Iron status biomarkers in iron deficient women consuming oily fish versus red meat diet

Specific recommendations for anemic individuals consist in increasing red meat intake, but the population at large is advised to reduce consumption of red meat and increase that of fish, in order to prevent the risk of developing cardiovascular disease. This study aimed to determine the effects of consuming an oily fish compared to a red meat diet on iron status in women with low iron stores. The study was designed attending the Consolidated Standards of Reporting Trials (CONSORT) statement guidelines. It was a randomised crossover dietary intervention study of two 8-week periods. Twenty-five young women with low iron stores completed the study. Two diets containing a total of 8 portions of fish, meat and poultry per week were designed differing only in their oily fish or red meat content (5 portions per week). At the beginning and the end of each period blood samples were taken and hemoglobin, hematocrit, serum ferritin, serum iron, serum transferrin, serum transferrin receptor-2 and the Zn-protoporphyrin/free-protoporphyrin ratio were determined. Food intake and body weight were monitored. During the oily fish diet, PUFA intake was significantly higher (p=0.010) and iron intake lower (mean±SD, 11.5±3.4 mg/dayvs. 13.9±0.1 mg/day, p=0.008), both diets providing lower mean daily iron intake than recommended for menstruating women. Although there were no significant differences after 16 weeks, serum ferritin moderately decreased and soluble transferrin receptor increased with the oily fish, while changes with the red meat diet were the opposite. In conclusion, an oily fish diet compared to a red meat diet does not decrease iron status after 8 weeks in iron deficient women.     

Effects of Hemin and Nitrite on Intestinal Tumorigenesis in the A/J Min/+ Mouse Model

Red and processed meats are considered risk factors for colorectal cancer (CRC); however, the underlying mechanisms are still unclear. One cause for the potential link between CRC and meat is the heme iron in red meat. Two pathways by which heme and CRC promotion may be linked have been suggested: fat peroxidation and N-nitrosation. In the present work we have used the novel A/J Min/+ mouse model to test the effects of dietary hemin (a model of red meat), and hemin in combination with nitrite (a model of processed meat) on intestinal tumorigenesis. Mice were fed a low Ca²⁺ and vitamin D semi-synthetic diet with added hemin and/or nitrite for 8 weeks post weaning, before termination followed by excision and examination of the intestinal tract. Our results indicate that dietary hemin decreased the number of colonic lesions in the A/J Min/+ mouse. However, our results also showed that the opposite occurred in the small intestine, where dietary hemin appeared to stimulate tumor growth. Furthermore, we find that nitrite, which did not have an effect in the colon, appeared to have a suppressive effect on tumor growth in the small intestine.     

Mechanism of colorectal carcinogenesis triggered by heme iron from red meat

Colorectal cancer (CRC) is one of the major tumor entities worldwide, with an increasing incidence in younger people. CRC formation is causally linked to various genetic, life-style and dietary risk factors. Among the ladder, the consumption of red meat has emerged as important risk factor contributing to CRC. A large body of evidence shows that heme iron is the critical component of red meat, which promotes colorectal carcinogenesis.In this review, we describe the uptake and cellular fate of both heme and inorganic iron in intestinal epithelial cells. Next, an overview on the DNA damaging properties of heme iron is provided, highlighting the DNA adducts relevant for CRC etiology. Moreover, heme triggered mechanisms leading to colonic hyperproliferation are presented, which are intimately linked to changes in the intestinal microbiota induced by heme. A special focus was set on the impact of heme iron on innate and adaptive immune cells, which could be relevant in the context of CRC. Finally, we recapitulate in vivo studies providing evidence for the tumor-promoting potential of dietary heme iron. Altogether, heme iron affects numerous key pathways involved in the pathogenesis of CRC.     

Adenosine 3',5'-monophosphate formation by preparations of rat liver soluble guanylate cyclase activated with nitric oxide, nitrosyl ferroheme, S-nitrosothiols, and other nitroso compounds

Cyclic AMP formation from ATP was stimulated by unpurified and partially purified soluble hepatic guanylate cyclase in the presence of nitric oxide (NO) or compounds containing a nitroso moiety such as nitroprusside, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), nitrosyl ferroheme, and S-nitrosothiols. Cyclic AMP formation was undetectable in the absence of NO or nitroso compounds and was not stimulated by fluoride or glucagon, indicating the absence of adenylate cyclase activity. The nitroso compounds failed to activate, whereas fluoride or glucagon activated, adenylate cyclase in washed rat liver membrane fractions. Cyclic GMP formation from GTP was markedly stimulated by the soluble hepatic fraction in the presence of NO or nitroso compounds. Cyclic AMP formation by partially purified guanylate cyclase was competitively inhibited by GTP and cyclic GMP formation is well-known to be competitively inhibited by ATP. Therefore, it appears that activated guanylate cyclase, rather than adenylate cyclase, was responsible for the formation of cyclic AMP from ATP. Formation of cyclic AMP of cyclic GMP was enhanced by thiols, inhibited by hemoproteins and oxidants, and required the addition of either Mg2+ or Mn2+. Further, several nitrosyl ferroheme compounds and S-nitrosothiols stimulated the formation of both cyclic AMP and cyclic GMP by the soluble hepatic fraction. These observations support the view that soluble guanylate cyclase is capable, under certain well-defined conditions, of catalyzing the conversion of ATP to cyclic AMP.     

Westernized high-fat diet accelerates weight loss in dextran sulfate sodium-induced colitis in mice,which is further aggravated by supplementation of heme

The Western diet, rich in fat and red meat, predisposes for inflammatory bowel disease (IBD); however, little is known about mechanisms involved. Red meat contains high levels of heme, a well-known inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1). Pharmacological induction of HO-1 ameliorates experimental colitis. We analyzed the effect of a westernized high-fat (HF) diet supplemented with heme on intestinal HO-1 expression and dextran sulfate sodium (DSS)-induced colitis.Mice were fed chow or HF diets for 2 weeks. In the second week, the HF diet was supplemented with or without 0.5 μmol/g heme. Subsequently, the 3 diet groups were given drinking water with or without 4% DSS to induce colitis.Significant body weight reduction was first observed after 4 days in the chow/DSS mice (?5±3%), whereas this was evident already after 2 days (?6±2%) in HF/DSS mice, showing increased weight loss compared to chow/DSS mice in the following days. Heme supplementation further aggravated DSS-induced weight loss in HF mice (?18±4% vs. ?7±5% for HF+heme/DSS vs. HF/DSS, P<.01). Heme increased HO-1 expression in the colon epithelium but decreased villin messenger RNA levels, indicating epithelial damage. In contrast, heme did not affect DSS-induced colon shortening and histological scores of epithelial damage and inflammation.A westernized diet accelerates DSS-induced weight loss in mice, which is further aggravated by heme, despite the induction of HO-1 in the colon epithelium. Our data warrant a detailed analysis of the association of (red) meat-containing diets and the development of IBD.     

Protein,iron, and meat consumption and risk for rheumatoid arthritis: a prospective cohort study

A recent prospective study showed that higher consumption of red meat and total protein was associated with increased risk for inflammatory polyarthritis. We therefore prospectively examined the relationship between diet (in particular, protein, iron, and corresponding food sources) and incident rheumatoid arthritis (RA) among 82,063 women in the Nurses' Health Study. From 1980 to 2002, 546 incident cases of RA were confirmed by a connective tissue disease screening questionnaire and medical record review for American College of Rheumatology criteria for RA. Diet was assessed at baseline in 1980 and five additional times during follow up. We conducted Cox proportional hazards analyses to calculate the rate ratio of RA associated with intakes of protein (total, animal, and vegetable) and iron (total, dietary, from supplements, and heme iron) and their primary food sources, adjusting for age, smoking, body mass index, and reproductive factors. The multivariate models revealed no association between RA and any measure of protein or iron intake. In comparisons of highest with lowest quintiles of intake, the rate ratio for total protein was 1.17 (95% confidence interval 0.89–1.54; P for trend = 0.11) and for total iron it was 1.04 (95% confidence interval 0.77–1.41; P for trend = 0.82). Red meat, poultry, and fish were also not associated with RA risk. We were unable to confirm that there is an association between protein or meat and risk for RA in this large female cohort. Iron was also not associated with RA in this cohort.     

Influence of supplemental high molecular weight pullulan or gamma-cyclodextrin on ileal and total tract nutrient digestibility, fecal characteristics, and microbial populations in the dog

This study was conducted to determine if supplemental pullulan and gamma-cyclodextrin affect canine nutrient digestibility, microbial populations, and fecal characteristics. Ileal cannulated dogs were fed a commercial diet, and treatments were administered daily in a 5 x 5 Latin square design: (i) no supplement; (ii) 2 g pullulan; (iii) 4 g pullulan; (iv) 2 g gamma-cyclodextrin; (v) 4 g gamma-cyclodextrin. Ileal and fecal samples were collected the last 4 d of each 14-d period. Increasing pullulan tended (p < 0.10) to linearly increase ileal bifidobacteria and lactobacilli and quadratically increase fecal lactobacilli. A similar response was noted in ileal bifidobacteria and lactobacilli with gamma-cyclodextrin. Gamma-Cyclodextrin resulted in a quadratic decrease (p < 0.05) in fecal Clostridium perfringens. Increasing pullulan linearly increased (p < 0.05) fecal score, while gamma-cyclodextrin resulted in a linear decrease (p < 0.05). Pullulan and gamma-cyclodextrin supplementation may have beneficial effects on the microbial ecology of dogs.     

Crystal structures of ferrous horse heart myoglobin complexed with nitric oxide and nitrosoethane

The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO-containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua-metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 A. The Fe-N-O angle of 147 degrees in hh MbNO is larger than the corresponding 112 degrees angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352-356) but is similar to the 150 degrees angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827-10836). The Fe-N(O) bond length of 2.0 A (this work) is longer than the 1.75 A distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 A from the imidazole N(epsilon) atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 A. The nitroso O atom of the EtNO ligand is located 2.7 A from the imidazole N(epsilon) atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein.     

Free radicals and the etiology of colon cancer

This hypothesis paper reviews diverse evidence suggesting that intracolonic production of oxygen radicals may play a role in carcinogenesis. The hypothesis began to evolve when the author made the chance discovery that 1/10,000 dilutions of feces generated detectable quantities of highly reactive hydroxyl radicals (HO.). The rate of HO. formation, detected using DMSO as a molecular probe, was quite remarkable, corresponding to that which would be produced by over 10,000 rads of gamma irradiation per day, absorbed in the periphery of the fecal mass adjacent to the mucosa. The relatively high concentrations of iron in feces, together with the ability of bile pigments to act as iron chelators that support Fenton chemistry, may very well permit efficient HO. generation from superoxide and hydrogen peroxide produced by bacterial metabolism. Such free radical generation in feces could provide a missing link in our understanding of the etiology of colon cancer: the oxidation of procarcinogens either by fecal HO., or by secondary peroxyl radicals (ROO.) to form active carcinogens or mitogenic tumor promotors. Intracolonic free radical formation may explain the high incidence of cancer in the colon and rectum, compared to other regions of the GI tract, as well as the observed correlations of a higher incidence of colon cancer with red meat in the diet, which increases stool iron, and with excessive fat in the diet, which may increase the fecal content of procarcinogens and bile pigments.     

Metabolic epidemiology of large bowel cancer. Fecal mutagens in high- and low-risk population for colon cancer. A preliminary report

Because of potential significance of fecal mutagens in the pathogenesis of colon cancer, the dietary pattern and fecal mutagens of 3 populations with distinct risk for the development of colon cancer, a high-risk population in New York Metropolitan area (non-Seventh-Day Adventists), a low-risk population of vegetarian Seventh-Day Adventists in New York Metropolitan area and a low-risk population in rural Kuopio, Finland were studied. The average daily intake of protein was the same in the 3 groups, but the sources were different, a greater portion coming from meat in the New York non-Seventh-Day Adventists and from vegetables in Seventh-Day Adventists. The intake of fat was lower in Seventh-Day Adventists and higher in Kuopio and in New York non-Seventh-Day Adventists. The intake of dietary fiber was high in Kuopio compared to other groups. Fecal samples collected for 2 days were freeze-dried extracted with peroxide-free diethyl ether, partially purified on a silica-gel column and assayed for mutagenicity using the Salmonella/mammalian microsome mutagenicity test. The mutagenic activity was observed with Salmonella typhimurium tester strain TA98 without microsomal activation and with TA100 with and without microsomal activation in high-risk subjects from New York consuming a high-fat, high-meat diet. The incidence of fecal mutagen activity was higher in volunteers from New York consuming a high-fat, high-meat diet compared to low-risk rural Kuopio population. None of the vegetarian Seventh-Day Adventists showed any mutagenic activity.     

Concentrations and antioxidative activity of anserine and carnosine in poultry meat extracts treated with demineralization and papain

Anserine and carnosine found in animal skeletal muscle are capable of inhibiting the catalysis of lipid oxidation by heme and non-heme iron. A demineralization technique and a proteolytic enzyme (papain) were used in this research in order to reduce the levels of proxidants while maintaining high levels of anserine and camosine in poultry (chicken, duck and turkey) meat extracts. Undemineralized poultry meat extracts contained larger amounts of anserine, camosine, heme and non-heme iron (p < 0.05) than did demineralized poultry meat extracts. Both undemineralized and demineralized breast meat extracts of chicken, duck and turkey contained higher concentrations of anserine and camosine, but lower amounts of heme and non-heme iron than did thigh meat extracts. In chicken, duck and turkey meat (breast and thigh) extracts (undemineralized and demineralized), the anserine concentrations were greater (p < 0.05) than the camosine concentrations. The hydrogen-donating ability of undemineralized and demineralized chicken breast meat extracts was not significantly different (p > 0.05): however, demineralized chicken breast meat extracts showed higher (p < 0.05) ferrous chelating ability than did undemineralized meat extracts. The concentrations of anserine, camosine, heme and non-heme iron in chicken breast meat extracts increased (p < 0.05) with the addition of papain (1%) to the meat mixture before extraction. Heme and non-heme iron in the chicken breast meat extracts increased as the reaction time for papain increased from 30 to 120 min, but the concentrations of anserine and camosine were not significantly affected by the longer reaction time for papain. The hydrogen-donating ability and ferrous chelating ability of demineralized chicken breast meat extracts were not significantly affected by papain. The ratios of carnosine/anserine were very specific in the chicken, duck and turkey meat extracts (breast and thigh); and the turkey meat extracts had lower (p < 0.05) camosine/anserine ratios than did the chicken and duck meat extracts. The camosine/anserine ratios of undemineralized and demineralized poultry meat extracts were not significantly different (p > 0.05). This suggests that the carnosine/anserine ratios of undemineralized chicken (0.62 - 0.80), duck (0.75 - 0.77) and turkey (0.15 - 0.16) meat extracts could be used to estimate the single meat species in uncooked or cooked meat products.     

Features of the metabolism of nitric oxide in normal state and inflammation

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes that hampered the progress of these studies were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several μM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was considered previously.     

S-nitrosohemoglobin: a mechanism for its formation in conjunction with nitrite reduction by deoxyhemoglobin.

The formation of S-nitrosohemoglobin (SNOHb) in red cells has been a major point of contention among researchers in this field. We have delineated a new mechanism for the formation of SNOHb coupled to nitrite reduction by deoxygenated hemoglobin chains at low oxygen pressures. The establishment of this mechanism required the development of a chemiluminescence assay utilizing Cu(II) and ascorbic acid to directly measure nitrosothiols without any interference from nitrite or heme-NO. The formation of SNOHb was shown to involve a dominant nitrite-reduction intermediate with electron delocalized between the heme iron and the bound NO. The possible mechanisms for the formation of SNOHb from this intermediate in the absence of oxygen are discussed including the role for an expansion of the electron delocalized intermediate to include the beta-93 cysteine residue. This extended delocalization was supported by a direct reaction with unbound NO, simultaneously producing SNOHb and Hb(II)NO, when NO reacts with metHb. The SNOHb found in red cells in vivo can, thus, be explained as originating from nitrite reduction that takes place at reduced oxygen pressures.     

Dietary Heme Alters Microbiota and Mucosa of Mouse Colon without Functional Changes in Host-Microbe Cross-Talk

Colon cancer is a major cause of cancer deaths in Western countries and is associated with diets high in red meat. Heme, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents which injures surface cells leading to compensatory hyperproliferation of crypt cells. This hyperproliferation results in epithelial hyperplasia which increases the risk of colon cancer. In humans, a high red-meat diet increases Bacteroides spp in feces. Therefore, we simultaneously investigated the effects of dietary heme on colonic microbiota and on the host mucosa of mice. Whole genome microarrays showed that heme injured the colonic surface epithelium and induced hyperproliferation by changing the surface to crypt signaling. Using 16S rRNA phylogenetic microarrays, we investigated whether bacteria play a role in this changed signaling. Heme increased Bacteroidetes and decreased Firmicutes in colonic contents. This shift was most likely caused by a selective susceptibility of Gram-positive bacteria to heme cytotoxic fecal water, which is not observed for Gram-negative bacteria, allowing expansion of the Gram-negative community. The increased amount of Gram-negative bacteria most probably increased LPS exposure to colonocytes, however, there is no appreciable immune response detected in the heme-fed mice. There was no functional change in the sensing of the bacteria by the mucosa, as changes in inflammation pathways and Toll- like receptor signaling were not detected. This unaltered host-microbe cross-talk indicates that the changes in microbiota did not play a causal role in the observed hyperproliferation and hyperplasia.     

Influence of diet on iron, copper, and zinc status in children under 24 months of age

The objective of the study was to determine whether iron and micronutrient status is improved with an increased amount of meat in the diet. To this end, a longitudinal prospective study with infants recruited at 4 mo and followed until 24 mo of age was undertaken. One hundred ninety-eight infants formed the original study cohort; 48 withdrew before the end of the study. Subjects were classified as nonmeat eaters or as mixed (red and white)-meat eaters subgrouped into tertiles depending on the meat content reported in diet diaries. Seven-day weighed food records were recorded at 4, 8, 12, 16, 20, and 24 mo. Blood samples taken at 4, 12, and 24 mo were analyzed for parameters of iron and micronutrient status. Iron intake increased during the first year, thereafter remaining constant. The percentages of subjects with hemoglobin values below 110 g/L were 34.1, 23.1, and 13.4 at 4, 12, and 24 mo, respectively. For parameters of iron status, the number of results below the reference range was determined for each diet group and a significant negative relationship between serum iron and meat intake at 12 mo of age was seen (p<0.023). There was a trend for hemoglobin concentrations to be inversely related to the meat intake, at the same age (p<0.068). No effects on zinc or copper status were seen. We conclude that a weak association between dietary meat and iron/Hb suggests a positive role for red meat. There was no disadvantage to the nonmeat-eating infants with respect to zinc or copper.     

Susceptibility of human metabolic phenotypes to dietary modulation

Dietary composition has been shown to influence metabolism and to impact on the prevalence and risk for certain diseases, but hitherto, there have been no systematic studies on the effects of dietary modulation of human metabolic phenotype (metabotype). Here, we have applied 1H NMR spectroscopy in combination with multivariate statistical analysis to characterize the effects of three diets: "vegetarian", "low meat", and "high meat" on the metabotype signature of human participants. Twelve healthy male participants (age range of 25-74 years) consumed each of these diets, in a randomized order, for continuous 15-day-periods with an intervening washout period between each diet of 7 days duration. Each participant provided three consecutive 24-hour urine collections on days 13, 14, and 15 of each dietary period, and 1H NMR spectra were acquired on all samples. Pattern recognition analysis allowed differentiation of the characteristic metabolic signatures of the diets with creatine, carnitine, acetylcarnitine, and trimethylamine-N-oxide (TMAO) being elevated in the high-meat consumption period. Application of orthogonal projection to latent structure discriminant analysis (O-PLS-DA) allowed the low-meat diet and vegetarian diet signatures to be characterized, and p-hydroxyphenylacetate (a microbial mammalian cometabolite) was higher in the vegetarian than meat diet samples, signaling an alteration of the bacterial composition or metabolism in response to diet. This work shows the potential for the routine use of metabonomics in nutritional and epidemiological studies, in characterizing and predicting the metabolic effects and the influence of diet on human metabotypes.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

Nitrite and nitroso compounds can serve as specific catalase inhibitors

Objective: We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness.

Methods: Catalase activity was evaluated by the permanganatometric and calorimetric assays.

Results: This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide.

Discussion: We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO⁺ group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.     

Resonance Raman and EPR of nitrosyl human hemoglobin and chains, carp hemoglobin, and model compounds. Implications for the nitrosyl heme coordination state.

We report the joint resonance Raman (RR) and electron paramagnetic resonance (epr) study of five- and six-coordinate nitrosyl heme model compounds and of the titled nitrosyl hemoproteins. Both epr and RR spectra fall into two types which, in the models, correspond to five- and six-coordinate nitrosyl hemes. However, neither RR nor epr spectroscopy is highly sensitive to the nature of the bond between a nitrosyl heme and a coordinated nitrogenous base, nor do the results of one technique uniformly correlate with those of the other. It is not possible to use epr spectroscopy as a test for the coordination state of a nitrosyl heme. The position of the highest frequency (depolarized) RR band possibly provides such a test. Any breaking of the very weak bond between nitrosyl heme and proximal histidine in T state human HbNO is more a consequence of tertiary structural features unique to the human alphaNO chains than it is of properties of the T quaternary conformation.