Two-Electron Reduction of Heme and Initiation of Heme Degradation

This is one possible psychedelic "loser" mechanism for the degradation of heme in association with the formation of a glutathionyl adduct. The "mechanism" is an adaptation, for better or worse, of the mechanisms proposed by these authors [Nakamura et al., 1984: (http://www.jbc.org/content/259/11/7080.full.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/6725282); Atamna and Ginsburg, 1995: (http://www.jbc.org/cgi/content/full/270/42/24876); Schaefer et al., 1985: (http://www.ncbi.nlm.nih.gov/pubmed/3927975)], and the main interest I have is in keeping track of the two-electron reduction of heme. It's difficult to keep track of things. It seems more likely that a thioperoxyl derivative of glutathione, as discussed by Atamna and Ginsburg (1995), would initiate the degradation reactions, but thioperoxyl species mimic the actions of peroxides, etc., and doing those again could get repetitive. Schaefer et al. (1985) proposed that the reaction would repeat more than once and produce dipyrroles and maleimides (or, here, related sulfhydryl compounds or disulfide cross-links with amino acid residues on proteins, etc.):

Note on the Reduction of Ferryl Heme by Hydrogen Peroxide

I was meaning to change this diagram to show the product that would be expected (the superoxide anion):

The point is that there's thought to be an unknown mechanism by which that superoxide can donate another electron to iron(IV), in ferryl heme in some peroxidase enzymes, to produce molecular oxygen and water, which I've drawn as one of the coordinating ligands. I should draw the reduction mechanism in the case of the hydrogen abstraction from an unsaturated fatty acid [RH ---> R(e-) + H(e-) (a hydrogen atom and an alkyl radical, R(e-), that can go on to form a lipid hydroperoxide and then a lipid alkoxyl radical, etc.)]. That's the overall reaction in the reduction of ferryl heme by hydrogen peroxide--a hydrogen abstraction (shown below). It makes more sense in view of that article by Traylor et al. (1993), cited in the previous posting. The reaction is very similar to an epoxidation, and Traylor et al. (1993) used different hemes to form epoxides in high yields. But ferryl heme doesn't normally form epoxides in vivo. They had to use special conditions. Incidentally, a hydrogen abstraction isn't the same as a hydride transfer:

Reductions of Ferryl Heme by Hydroperoxides; Reduction of Perferryl Heme via the Oxidation of a Tyrosine Residue; Heme-to-Protein Cross-Linking

It helps me to go through these reactions and to thereby avoid "having" to make use of these types of diagrams or equations in other contexts. If I can understand these things, I won't have to catalog equations, as so many articles do, or even discuss most of these things. One reason that many of the articles are so confusing is that hydrogen peroxide or an organic hydroperoxide (or a protein "substrate") can reduce ferryl hemes to ferric hemes or perferryl hemes to ferryl hemes. Those same compounds usually act as oxidants, however, and they also oxidize ferric heme to perferryl heme or ferrous to ferryl hemes. But anyway, the reduction of ferryl heme by HOOH (hydrogen peroxide) is more or less the same reaction as the reduction by an organic hydroperoxide, such as linoleate hydroperoxide or another lipid hydroperoxide. I'm not showing the reduction of perferryl to ferric heme, but that's the reaction that allows for the true "peroxidase" activity of non-protein-bound heme (or at least that's one way of looking at it). The products are O2, water, and ferric heme, even though there's a superoxo-Fe(III)-heme "intermediate." That's supposedly one reason some peroxidase enzymes can exert antioxidant effects at some low-to-intermediate concentrations. There's an intermediate range at which the peroxide-to-protein molar ratio allows for the consumption of hydrogen peroxide, by allowing the perferryl-to-ferric reduction pathway to predominate over the more damaging ferryl-to-ferric reduction pathway, but that doesn't usually work out too well in vivo, seemingly. The reaction I'm showing for the one-electron reduction of ferryl heme (one proposed by Traylor and Xu, 1987, cited below) by HOOH is similar, however, to that perferryl-to-ferric pathway. The reduction of ferryl to ferric heme is thought to produce a perhydroxyl species [Nagababu and Rifkind, 2004: (http://www.ncbi.nlm.nih.gov/pubmed/15548894)] or a peroxyl radical, etc. (that's the reason it's more damaging, under some conditions, than the other reduction pathway). The mechanisms aren't known (Traylor et al., 1993, cited below). Anyway, some antioxidants are likely to act by very similar one-electron reduction mechanisms. These two are adapted from the mechanisms proposed by Traylor et al. (1993) [Traylor et al., 1993: (http://pubs.acs.org/doi/abs/10.1021/ja00060a027)] and other authors [Traylor and Xu, 1987: (http://pubs.acs.org/doi/abs/10.1021/ja00254a059); Schaefer et al., 1985: (http://www.ncbi.nlm.nih.gov/pubmed/3927975)] and show the one-electron reduction of ferryl heme by an organic hydroperoxide, yielding ferric heme and an alkoxyl radical [the ROO radical shown, where R = a lipid or protein or other substrate, such as some antioxidants (ROO could also be RNO in some cases, etc.)] and, below that, a "hack" mechanism for the one-electron reduction of ferryl heme (to ferric heme) by hydrogen peroxide.

This is an adaptation of the mechanism proposed by Chu et al. (2000) for the formation of a tyrosyl phenoxyl radical via the reduction of a copper(II)-amine complex [Chu et al., 2000: (http://pubs.acs.org/cgi-bin/jtext?jpcbfk/104/i15/abs/jp994487d)(http://www.chem.yorku.ca/profs/hopkin/87.pdf)].
This shows the oxidation of a tyrosine residue by perferryl heme, and the reaction transfers the porphyrin radical and the "cation" of perferryl heme, in effect, to two different amino acid residues of a protein. This is known to occur to some extent, and the radical is delocalized throughout many amino acid residues of the protein, etc.

This is a hypothetical, proposed "bookkeeping" mechanism and shows a two-electron reduction
of perferryl heme to ferric heme that forms an intramolecular glycol and a heme-protein cross-link on one of the bridgehead carbons, I guess one would say:

Two-Electron Oxidation of Ferric Heme to "Perferryl" Heme: Cleavage of the Dioxygen Bond is Normally Heterolytic in Protic Solvents, for God's Sake

I'm basically putting these up for my own benefit and to see if it's possible to see them on Google's "2-inch-wide" blog space. It helps me to keep track of the "bookkeeping" of the electron distributions and formal charges to do this, even though perferryl heme has a net charge of 0, as far as I can tell, in the physiological pH range (and even though this is unlikely to be of interest to anyone). This type of thing is more like "meditation" than anything else, and doing it lets me use the obnoxious drawing program. The wireless is down, and, as a result, I'm "grounded" in nerd tasks.

The mechanism for the formation of perferryl heme {a.k.a. "Compound I," an intermediate that forms during the enzymatic cycling of ferryl and ferric heme, in peroxidase and catalase enzymes and in cytochrome P450-containing enzymes, and that also forms during the ferric-ferryl cycling of heme in aqueous solutions of non-protein-bound, monomeric or dimeric heme [initially as hemin (chloro-coordinated heme) & hematin (hydroxo-coordinated Fe(III), etc.)]} is my adaptation (meaning a "loser" electron-bookkeeping venture that makes sense) of the intramolecular mechanism proposed by Schaefer et al. (1985) [Schaefer et al., 1985: (http://www.ncbi.nlm.nih.gov/pubmed/3927975)] for the formation of a glycol, as an intermediate species in the degradation of heme to "oligopyrroles" and maleimides, and a cation radical (such that the electron density is "localized," in the porphyrin radical that's delocalized across the entire ring system, primarily on the meso carbons and pyrollic nitrogens, as it's supposed to be [La Mar et al., 1981: (http://www.jbc.org/cgi/reprint/256/1/237.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/7451437)]) and the mechanisms proposed by Ozaki et al. (2001) [Ozaki et al., 2001: (http://pubs.acs.org/doi/abs/10.1021/ar9502590)]. Perferryl heme, or Fe(V)-heme, is actually Fe(IV)-heme with a porphyrin radical, and researchers don't use consistent forms of notation in their descriptions of it as being either a pentavalent species or a tetravalent species with a pi-cation radical on the porphyrin ring. It's tetravalent, though, and it's almost impossible for me to make sense of the reactions without doing some electron bookkeeping and getting a crude understanding of the electron distribution in the different species.

I've shown a way of viewing the formation of the porphyrin radical that helps me understand the electron donation. One electron of the iron-oxygen "double bond," which is really a mess of complex d(pi)-p(pi) interactions and d(pi)-p*(pi), or whatever, backbonding interactions (it's basically intramolecular hyperconjugation that they're talking about, as far as I can tell), comes from iron, and one comes from the porphyrin ring, from a bookkeeping standpoint. The bond order is larger than 2 for the ferryl (it's analogous to a "carbonyl," C=O, group) oxygen-iron bond. In any case, it's a two-electron oxidation of iron and of the porphyrin ring, as a whole, from a formal-charge-sort-of oxidation state of Fe(3+) to Fe(5+), even though the heme iron is hexacoordinated or maybe pentacoordinated in the physiological pH range and is likely to be neutral, except in the "crypto-" protonated ferryl and perferryl heme species, in which the net charge is +1 [Carlsen et al., 2005: (http://cat.inist.fr/?aModele=afficheN&cpsidt=16551176)]. The site(s) of protonation are unknown but are near the heme iron in an unidentified location (Carlsen et al., 2005) [maybe on the nitrogens and the oxygen, rapidly exchanged by proton transfers in perferryl heme (a.k.a. compound I)? (http://scholar.google.com/scholar?hl=en&q=%22proton+transfer%22+nitrogen+pyrrolic+heme+cation+radical&as_ylo=&as_vis=0)]. I say the oxygen because there's evidence that the ferryl oxygen can be protonated at physiological pH values in some chloroperoxidase enzymes [Newcomb et al., 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18174331)]. Damn, that's exciting--"basic ferryl groups" (Newcomb et al., 2008, p. 8179) on some heme-containing enzymes. But one way of looking at the internal electron transfer that forms perferryl heme, as shown in Figure 11 of Schaefer et al. (1985) and Figure 1 of Pan et al. (2006) [Pan et al., 2006: (http://www.ncbi.nlm.nih.gov/pubmed/16500709)], is to view pentavalent heme as being an "intermediate" or resonance form that doesn't really exist as a distinct species or that involves an abstraction, in another step that doesn't really work that way, of a second electron from the porphyrin ring.

I've shown the heterolytic cleavage of the dioxygen bond of an organic hydroperoxide (RO-OH of ROOH), and the upshot of a lot of articles is that the cleavage of that bond is heterolytic and not homolytic, under most conditions (except in the presence of micellar aggregates, to some extent). But lipid hydroperoxides (LOOH) and other ROOH compounds can still be oxidized by ferryl heme species to form radical products (Traylor et al., 1993: (http://pubs.acs.org/doi/abs/10.1021/ja00060a027)]. I'm showing the oxidation of ferric heme (to perferryl heme) by an ROOH species (this includes hydrogen peroxide, or HOOH). I'm too tired to upload my proposed mechanism for the transfer of a cation radical from the porphyrin ring to a tyrosine residue, to form a tyrosyl phenoxyl radical. Why not save some fun for another day.

Here's the initial step, showing the oxidation of ferric heme by an organic hydroperoxide or hydrogen peroxide, and the participation of or "catalysis" by a generic base, B(-):

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Trick for Overriding "Stemming" on Google Scholar

This is a not-very-advanced trick for overriding the "stemming" feature that can sometimes become problematic in searches on google scholar. In some cases, google scholar automatically stems words, such as creatine, to include the word creatinine, and that can give excessive numbers of results. I've seen other examples of search terms that produce that kind of "garbage" in the search results, but I can't think of those terms now. The big secret is to put the single word in quotations. This search gives a lot of junk on creatinine (http://scholar.google.com/scholar?hl=en&q=turnover+export+creatine) and provides 8,640 results, but this search, in which I meant to find articles discussing the mechanisms governing the export of creatine from cells and, hence, the total, intracellular creatine+phosphocreatine pools in cells, gave 3,860 results (http://scholar.google.com/scholar?hl=en&q=turnover+export+%22creatine%22) and less junk.

Magnesium as a CCK-Releasing Stimulus: Potential Relevance to the Use of Magnesium as an Adjunctive Treatment in Liver Disease

In this article [Ko, 2008: (http://www.ncbi.nlm.nih.gov/pubmed/18289201)], Ko (2008) discussed this article (the article the editorial accompanies) [Tsai et al., 2008: (http://socgastro.com/biblioteca/rev-articulos/pdf/magnesio200802.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/18076730)]. Tsai et al. (2008) found that higher intakes of dietary magnesium (Mg2+) intakes were associated with a decrease in the risk of developing symptomatic gallstones. I guess they looked at men only (?). It's not just relevant to men, of course. But Ko et al. (2008) cited some of that old research showing that intraduodenal Mg2+, in the form of Mg2+ salts and not chelates, induces gallbladder contractions by inducing cholecystokinin CCK release (http://scholar.google.com/scholar?hl=en&q=cholecystokinin+magnesium+gallbladder) from, I guess, enterocytes. I haven't read enough of all that research to get a sense of the mechanism, but the site of action is apparently localized. They've used large doses of Mg2+ in some of those articles, but I did some quick searching and reading and found a dose-response study in dogs that would tend to argue against the need for massive dosages. Ko (2008) was basically saying that normal amounts of Mg2+ could exert the same effect on a smaller scale.

On the surface, that research doesn't look like much, and the same effect can supposedly be shown for other electrolytes. I don't know, though. This article [Colombato et al., 1977: (http://www.ncbi.nlm.nih.gov/pubmed/835560)] shows a 17-fold increase in the rate of bilirubin output from the gallbladder/common bile duct in normal people, in response to Mg2+. That's significant, but the main thing I was thinking, in view of that editorial by Ko (2008), is that changes in the relative abundances of bile acids or increases in the biliary and intestinal-luminal-fluid concentrations of unconjugated bilirubin occur in people with liver disease and can bind massive amounts of magnesium [see here for some articles: (http://hardcorephysiologyfun.blogspot.com/2009/09/heterogeneous-precipitationnucleation.html)]. In one of those articles in the past posting [Heubi et al., 1997: (http://www.ncbi.nlm.nih.gov/pubmed/9285381)], the authors found that the equivalent of an adult's intake of 1100-2300 mg of Mg2+ per day was necessary to just prevent overt Mg2+ deficiency in children with liver disease, and that was attributed mainly to the sequestration of Mg2+ in the GI tract. In any case, in theory, that Mg2+ would exist mainly as mixed precipitates, bound to free fatty acids or bile salts or bilirubin, etc., and wouldn't be capable of participating in osmoregulatory disturbances. So the people might be able to tolerate much higher dosages of Mg2+ than people who do not have liver disease would. Some authors have remarked on the drastic differences in the amounts of Mg2+ that people can tolerate. That type of precipitation could be one reason for the large amount of variation. I think Ko (2008) also mentioned the anti-nucleating effect that Mg2+ could have. Magnesium binds to bilirubin or otherwise interacts with it in complex ways, also (http://scholar.google.com/scholar?hl=en&q=magnesium+bilirubin+binding), and can apparently prevent bilirubin from binding to precipitated calcium phosphate [Van der Veere et al., 1995: (http://www.jlr.org/cgi/reprint/36/8/1697.pdf)(http://www.ncbi.nlm.nih.gov/pubmed/7595091)]. In general, Mg2+ has an "anti-nucleating" effect, in different contexts, even though I've seen articles discussing the capacity of Mg2+ to promote amorphous calcium phosphate formation but to also help to prevent, to a significant degree, soft-tissue hydroxyapatite/apatite crystallization. Amorphous calcium phosphate is basically innocuous, as far as I know, particularly in comparison to hydroxyapatite (in soft tissues). Anyway, the point is that people who have liver disease could require large amounts of Mg2+ to just to prevent deficiency (there's a lot of research showing that Mg2+ depletion contributes to exercise intolerance in liver disease, etc.), and even larger dosages could be required to produce the CCK-releasing effects that would occur in normal people at lower dosages. They used to say that CCK suppresses the appetite and used to suggest that people use L-phenylalanine to release CCK, etc. I don't think that's a good idea, but, in view of the problematic quality of much of the research on Mg2+ (research that is plagued by the use of miniscule and arbitrary dosages of oral Mg2+ and massive dosages of intravenous Mg2+ for everything under the sun), I wouldn't be surprised if the regulation of gallbladder contraction by Mg2+ is another thing that people stopped paying attention to in the 1980's, after the Bayh-Dole Act was passed or something. Bayh and Dole told them to buy influence in Universities via special interest groups and dole out advertising money for endless tv ads, and they "Bayh-ed it." No, that's nonsense.

Dependence of Antiapoptotic Effects of Glutamine on de novo Uridine Biosynthesis

Evans et al. (2005) [Evans et al., 2005: (http://ajpgi.physiology.org/cgi/reprint/289/3/G388)(http://www.ncbi.nlm.nih.gov/pubmed/15878985?dopt=Abstract)] found that the antiapoptotic effects of physiological concentrations of glutamine (GLN) (500 uM) in cultured, colonic epithelial cells were mediated, at least in part, by the capacity of GLN to serve as a precursor for pyrimidine (uridine and the other pyrimidine nucleosides and nucleotides derived from it) biosynthesis. One thing that's interesting is that the GLN-induced maintenance of pyrimidine nucleotide concentrations did not depend on transcription or on DNA replication, and it's possible that the maintenance of the pyrimidine pool maintained glucose uptake by maintaining UDP-hexosamine-dependent glycosylation reactions, etc. These results don't mean that GLN didn't partially prevent apoptosis by serving as a precursor of 2-oxoglutarate and the TCA cycle intermediates that are formed from the entry of 2-oxoglutarate into the TCA cycle. The results just mean that the maintenance of a pyrimidine pool can be an obligatory condition for the prevention of apoptosis in response to certain apoptotic stimuli. The fact that inosine, at millimolar concentrations, could prevent apoptosis in those experiments shows that ribose-5-phosphate and hypoxanthine, derived from inosine, can help maintain the ATP and PRPP, etc., that are required for pyrimidine salvage and de novo pyrimidine formation and for other antiapoptotic effects.

One interpretation of that article is that glutamine, taken at excessive dosages in people who have chronic liver disease or other inflammatory conditions, could exacerbate liver disease or other disease processes that are characterized by impairments in mitochondrial functioning. Orotate, an intermediate in uridine biosynthesis, can be used to induce fatty liver disease in animals, in part by depleting ATP and purine nucleotide levels [see here: (http://hardcorephysiologyfun.blogspot.com/2009/08/some-more-old-papers-of-mine.html)]. GLN is a substrate of type II (cytosolic) carbamoyl phosphate synthase ["glutamine-utilizing carbamoyl phosphate synthase (CPS-II)], and there would mainly be an accumulation of cytosolic carbamoyl phosphate (CP) in people exhibiting impairments in the overall activity of the urea cycle, such as in chronic liver disease. In the context of a trauma or other acute injury that can increase glutamine turnover, there probably wouldn't be the kind of damage to the mitochondria that would cause mitochondrial CP to be exported to the cytosol and cause orotate formation to increase pathologically. But mitochondrial dysfunction could be expected to cause orotate and dihydroorotate to accumulate, given that dihydroorotate dehydrogenase is a mitochondrial enzyme whose activity can be impaired in mitochondrial disorders. Szondy and Newsholme (1989) [Szondy and Newsholme, 1989: (http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=1138925&blobtype=pdf)(http://www.ncbi.nlm.nih.gov/pubmed/2803258)] noted, however, that only something like 0.4 percent of the glutamine that's utililzed (the Km of GLN for glutamine utilization overall, by all pathways, is 2.5 mM) in proliferating lymphocytes goes toward pyrimidine biosynthesis, and the Km of glutamine for binding to CPS-II is 16 uM. That implies that the rate of pyrimidine biosynthesis is unlikely to be sensitive to increases in GLN availability. Also, Evans et al. (2005) used 500 uM GLN to prevent apoptosis, and the plasma GLN concentration and, hence, extracellular fluid GLN concentrations are around 400-600 uM in humans. But the regulation of pyrimidine biosynthesis may not always be normal or comparable in other tissues or individuals, and that's another reason not to use excessive dosages. Ammonia derived from GLN deamidation could also contribute to adverse effects in some cases, but one would mainly expect to see that in some disease states.