Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: Evidence for a novel oxidative pathway

Sugiyama, Katsumi; Highet, R. J.; Woods, Amina S.; Cotter, Robert J.; Osawa, Yoichi

doi:10.1073/pnas.94.3.796

Cited by 46 publications

(45 citation statements)

References 40 publications

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“…Analysis of heat-exposed protein results in additional ions including (heme ϩ 16 Da) and a host of other unidentified species (Figure 4b). This result strongly suggests that heme itself undergoes covalent modifications during heat exposure, an interpretation that is supported by studies on myoglobin under oxidizing conditions [75]. In addition to the modifications uncovered here, other sites of oxidation likely exist.…”

Section: Oxidative Modificationssupporting

confidence: 65%

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solutionphase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11ϩ, whereas a considerably lower degree of oxidation is observed for the 7ϩ and 8ϩ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high-and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein. A number of well-established tools are available for experiments of this kind, including X-ray crystallography, nuclear magnetic resonance, calorimetry, and various spectroscopic techniques. Electrospray ionization mass spectrometry (ESI-MS) has become another widely used approach for exploring the properties of proteins in solution, providing information complementary to that obtained by traditional methods [1]. The ESI process generates intact gas-phase ions from proteins in solution. In the commonly used positive-ion mode these [M ϩ nH] nϩ species are multiply charged because of proton attachment.ESI of natively folded proteins results in protonation states n similar to those predicted for water droplets of the same size that are charged to the Rayleigh limit. This finding supports the idea that ionization follows the charged-residue model [2-6], although alternative scenarios have been proposed as well [7][8][9]. Much higher protonation states and wider charge-state distributions are generally seen for proteins that are unfolded in solution. Based on this empirical relationship, ESI-MS is now routinely being used for monitoring structural transitions in response to changes in pH, temperature, organic co-solvents, or covalent modifications [1, 10 -16]. The physical basis for the striking dependence of...

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Section: Oxidative Modificationssupporting

confidence: 65%

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Liu

Konermann

2009

J. Am. Soc. Mass Spectrom.

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“…These haems not covalently bound to the protein (9). There is also some evidence of sodium and TFA adducts (e.g., compare m/z 17586 with 17701).…”

Section: Resultsmentioning

confidence: 98%

Histidine and not tyrosine is required for the Peroxide‐induced formation of haem to protein cross‐linked myoglobin

et al. 2007

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SummaryPeroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 -10541).Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr 103 ! Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome. IUBMB Life, 59: 477-489, 2007

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“…Heme Assay-The amount of heme was determined by HPLC as described previously (21). Samples were injected onto an HPLC column (C4 Vydac, 5 m, 0.21 ϫ 15 cm) equilibrated with solvent A (0.1% trifluoroacetic acid) at a flow rate of 0.3 ml/min.…”

Section: Methodsmentioning

confidence: 99%

“…Large amounts of albumin are present in the medium because it was added to stabilize the heme as a heme⅐protein conjugate. The formation of altered heme products occurs when hemoproteins, including heme⅐albumin conjugates (29), react with reactive intermediates, such as hydrogen peroxide (21,30). As shown in Fig.…”

Section: Both Geldanamycin and Radicicol Inhibit Heme-initiated Dimermentioning

confidence: 99%

hsp90 Is Required for Heme Binding and Activation of Apo-Neuronal Nitric-oxide Synthase

Billecke

Bender²,

Kanelakis

et al. 2002

Journal of Biological Chemistry

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It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 M radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic hemebinding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor geldanamycin uncouples endothelial NOS activity and increases endothelial NOSdependent O 2 . production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 M, geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of geldanamycin as a specific inhibitor of hsp90 in redox-active systems.The endothelial and neuronal isoforms of nitric-oxide synthase (NOS) 1 have been reported to exist in heterocomplexes with hsp90 (1, 2). These proteins join a list of numerous other signaling proteins, including steroid receptors, some transcription factors, and a variety of protein kinases, that are associated with and regulated by hsp90 (for a review, see Ref.3). These signaling protein⅐hsp90 heterocomplexes are assembled in an ATP-dependent process by a five-protein system in which hsp90 and hsp70 are essential assembly components and Hop, hsp40, and p23 function as non-essential co-chaperones (4). One of the most studied hsp90-bound proteins is the glucocorticoid receptor (GR), which must be associated with hsp90 to have steroid binding activity (5, 6). Hsp90 binds directly to the ligand-binding domain of the GR (3), and biochemical data (7) coupled with data from GR mutants (8, 9) support the notion (6) that formation of a complex with hsp90 opens up a hydrophobic pocket in the ligand-binding domain to access by steroid. We have proposed a similar model for neuronal NOS (nNOS) in which the hsp90-based chaperone machinery acts in vivo to open the heme-binding cleft in heme-deficient apo-nNOS to access by heme (2).In contrast to the observations with steroid receptors and nNOS, it has been proposed that hsp90 regulates endothelial NOS (eNOS) through an allosteric mechanism. GarciaCardeñ a et al.(1) were able to demonstrate direct activation of purified eNOS catalytic activity by purified hsp90 in the absence of ATP, hsp70, and th...

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Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: Evidence for a novel oxidative pathway

Abstract: ABSTRACT

Cited by 46 publications

References 40 publications

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

Histidine and not tyrosine is required for the Peroxide‐induced formation of haem to protein cross‐linked myoglobin

hsp90 Is Required for Heme Binding and Activation of Apo-Neuronal Nitric-oxide Synthase

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