Spectroscopic studies of myoglobin at low pH: heme structure and ligation

Sage, J. Timothy; Morikis, Dimitrios; Champion, P. M.

doi:10.1021/bi00219a010

Cited by 168 publications

(248 citation statements)

References 74 publications

(92 reference statements)

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“…It seems possible that even at high acetonitrile concentrations some residual heme-protein interactions persist [62]. Evidence for interactions of this kind in denatured Mb has previously been obtained by vibrational spectroscopy, albeit under different solvent conditions [65]. The ESI-MS data presented below indicate that weakly associated complexes between unfolded Mb and heme may also persist under the conditions of the current study.…”

Section: Optical Studies and Esi Mass Spectramentioning

confidence: 48%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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This study describes a novel approach for monitoring noncovalent interactions in solution by electrospray mass spectrometry (ESI-MS). The technique is based on measurements of analyte diffusion in solution. Diffusion coefficients of a target macromolecule and a potential low molecular weight binding partner are determined by measuring the spread of an initially sharp boundary between two solutions of different concentration in a laminar flow tube (Taylor dispersion), as described in Rapid Commun. Mass Spectrom. 2002Spectrom. , 16, 1454Spectrom. -1462. In the absence of noncovalent interactions, the measured ESI-MS dispersion profiles are expected to show a gradual transition for the macromolecule and a steep transition for the low molecular weight compound. However, if the two analytes form a noncovalent complex in solution the dispersion profiles of the two species will be very similar, since the translational diffusion of the small compound is determined by the slow Brownian motion of the macromolecule. In contrast to conventional ESI-MS-based techniques for studying noncovalent complexes, this approach does not rely on the preservation of solution-phase interactions in the gas phase. On the contrary, "harsh" conditions at the ion source are required to disrupt any potential gasphase interactions between the two species, such that their dispersion profiles can be monitored separately. The viability of this technique is demonstrated in studies on noncovalent heme-protein interactions in myoglobin. Tight noncovalent binding is observed in solutions of pH 10, both in the absence and in the presence of 30% acetonitrile. In contrast, a significant disruption of the noncovalent interactions is seen at an acetonitrile content of 50%. Under these conditions, the diffusion coefficient of heme in the presence of myoglobin is only slightly lower than that of heme in a protein-free solution. A breakdown of the noncovalent interactions is also observed in aqueous solution of pH 2.4, where myoglobin is known to adopt an acid-unfolded conformation. (J Am Soc Mass Spectrom 2003, 14, 430 -441)

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Section: Optical Studies and Esi Mass Spectramentioning

confidence: 48%

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Clark

Konermann

2003

J. Am. Soc. Mass Spectrom.

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“…The native heme-protein interactions are largely disrupted and the heme groups are mostly solventaccessible. Yet, the persistence of weak residual hemeprotein interactions under these non-native conditions is quite likely [66,73], which is supported by the observation of low intensity heme-bound species by ESI-MS (Figure 3c). Also, "free" heme would get lost during dialysis, thus precluding refolding of the protein.…”

Section: Characterization Of Denatured and Refolded Hb By Optical Spementioning

confidence: 60%

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

Boys

Konermann

2007

J. Am. Soc. Mass Spectrom.

101

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The native structure of hemoglobin (Hb) comprises two ␣-and two ␤-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded ␣-and ␤-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric ␣-and ␤-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded ␣-and ␤-globin. However, some unexpected features are observed, e.g., a heme-deficient ␤-globin dimer that represents an off-pathway intermediate. Monomeric ␤-globin is capable of binding heme before forming a complex with an ␣-subunit. This observation suggests that holo-␣-apo-␤ globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes. (J Am Soc Mass Spectrom 2007, 18, 8 -16)

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“…Bond formation impedes protonation, and consequently, a pHdependent change in the absorbance spectra with pK Ϸ 4.4 is absent. From acid denaturation studies of Mb it is known that the covalent bond between the proximal histidine and the heme iron breaks at pH Ͻ 3.5, with subsequent protonation of the histidine (37,38). A similar scenario is likely for Ngb involving both axial histidines.…”

Section: Resultsmentioning

confidence: 98%

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

Nienhaus

Kriegl

Nienhaus

2004

Journal of Biological Chemistry

132

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We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4 -11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His 64 and Lys 67 . His 64 interferes with exogenous ligand access to the heme iron. Lys 67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.

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Spectroscopic studies of myoglobin at low pH: heme structure and ligation

Cited by 168 publications

References 74 publications

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

Folding and assembly of hemoglobin monitored by electrospray mass spectrometry using an on-line dialysis system

Structural Dynamics in the Active Site of Murine Neuroglobin and Its Effects on Ligand Binding

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