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Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

Silaghi-Dumitrescu R, Svistunenko DA, Cioloboc D, Bischin C, Scurtu F, Cooper CE - Nitric Oxide (2014)

Bottom Line: We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin.The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution.The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

View Article: PubMed Central - PubMed

Affiliation: "Babeş-Bolyai" University, 1 Mihail Kogalniceanu str., RO-400084 Cluj-Napoca, Romania; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Electronic address: rsilaghi@chem.ubbcluj.ro.

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Potential energy surface for nitrite linkage isomerization on a model of ferric myo/hemoglobin (UBP86/6-31G**).
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f0015: Potential energy surface for nitrite linkage isomerization on a model of ferric myo/hemoglobin (UBP86/6-31G**).

Mentions: Nitrite binds to the reduced and oxidized forms of heme proteins and, in enzymes such as cytochrome cd1 nitrite reductase and cytochrome c nitrite reductase, undergoes reduction to nitric oxide or ammonia [1–9]. More recently, interest has been focused upon nitrite reduction by hemoglobin (Hb), a reaction which has been proposed to have medical/physiological relevance [10–16]. Nitrite has traditionally been observed to bind to the iron in hemes and hemoproteins via the nitrogen atom (leftmost structure in Fig. 1) [2]. Based on density functional theory (DFT) calculation, we have previously proposed that binding of nitrite to the heme iron via its oxygen atom should also be feasible (rightmost structure in Fig. 1) – hence a nitrite linkage isomerism phenomenon; we further argued that this previously ignored binding mode would have mechanistic and physiological relevance [2]. This hypothesis has since been partially confirmed experimentally by the crystal structures of myoglobin and hemoglobin with nitrite, which have identified nitrite bound to iron only via the oxygen atom[17–19]. From an experimental point of view, this finding places globins (only binding nitrite via oxygen) [18] and cytochrome reductases (only binding nitrite via nitrogen) [1]in contrast to each other; notably, the two linkage isomers have not been observed at the same time, in the same reaction mixture, with the same protein. Somewhat related to this issue, EPR spectra have recently been reported for ferric Hb-nitrite adducts, surprisingly showing a complete lack of signals attributable to nitrite-bound Hb [10]. This finding was reminiscent of the similar EPR silence of a nitrite adduct of ferric heme d1-nitrite adduct of the enzyme cytochrome cd1 nitrite reductase [4,20]. These EPR silence phenomena have received several possible explanations, none of which are to our knowledge generally accepted. Among these would be a significant degree of structural inhomogeneity, fast rotation of the nitrite ligand around the iron–nitrogen bond, or a so-called uniaxial state as detailed below [10,18,20]. On the other hand Singel and co-workers as well as Young and Siegel have reported that nitrite-hemoglobin adducts do show entirely detectable EPR signals, characterized by specific g-values at ~2.9 [20–22], while others have further shown that such signals show buffer dependence and can indeed appear almost non-detectable under certain conditions [15,23].


Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

Silaghi-Dumitrescu R, Svistunenko DA, Cioloboc D, Bischin C, Scurtu F, Cooper CE - Nitric Oxide (2014)

Potential energy surface for nitrite linkage isomerization on a model of ferric myo/hemoglobin (UBP86/6-31G**).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4256065&req=5

f0015: Potential energy surface for nitrite linkage isomerization on a model of ferric myo/hemoglobin (UBP86/6-31G**).
Mentions: Nitrite binds to the reduced and oxidized forms of heme proteins and, in enzymes such as cytochrome cd1 nitrite reductase and cytochrome c nitrite reductase, undergoes reduction to nitric oxide or ammonia [1–9]. More recently, interest has been focused upon nitrite reduction by hemoglobin (Hb), a reaction which has been proposed to have medical/physiological relevance [10–16]. Nitrite has traditionally been observed to bind to the iron in hemes and hemoproteins via the nitrogen atom (leftmost structure in Fig. 1) [2]. Based on density functional theory (DFT) calculation, we have previously proposed that binding of nitrite to the heme iron via its oxygen atom should also be feasible (rightmost structure in Fig. 1) – hence a nitrite linkage isomerism phenomenon; we further argued that this previously ignored binding mode would have mechanistic and physiological relevance [2]. This hypothesis has since been partially confirmed experimentally by the crystal structures of myoglobin and hemoglobin with nitrite, which have identified nitrite bound to iron only via the oxygen atom[17–19]. From an experimental point of view, this finding places globins (only binding nitrite via oxygen) [18] and cytochrome reductases (only binding nitrite via nitrogen) [1]in contrast to each other; notably, the two linkage isomers have not been observed at the same time, in the same reaction mixture, with the same protein. Somewhat related to this issue, EPR spectra have recently been reported for ferric Hb-nitrite adducts, surprisingly showing a complete lack of signals attributable to nitrite-bound Hb [10]. This finding was reminiscent of the similar EPR silence of a nitrite adduct of ferric heme d1-nitrite adduct of the enzyme cytochrome cd1 nitrite reductase [4,20]. These EPR silence phenomena have received several possible explanations, none of which are to our knowledge generally accepted. Among these would be a significant degree of structural inhomogeneity, fast rotation of the nitrite ligand around the iron–nitrogen bond, or a so-called uniaxial state as detailed below [10,18,20]. On the other hand Singel and co-workers as well as Young and Siegel have reported that nitrite-hemoglobin adducts do show entirely detectable EPR signals, characterized by specific g-values at ~2.9 [20–22], while others have further shown that such signals show buffer dependence and can indeed appear almost non-detectable under certain conditions [15,23].

Bottom Line: We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin.The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution.The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

View Article: PubMed Central - PubMed

Affiliation: "Babeş-Bolyai" University, 1 Mihail Kogalniceanu str., RO-400084 Cluj-Napoca, Romania; Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Electronic address: rsilaghi@chem.ubbcluj.ro.

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