Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: evidence for intramolecular reorientation about a heme two-fold axis

Mar, Gerd N. La; Toi, Hiroo; Krishnamoorthi, Ramaswamy

doi:10.1021/ja00333a050

Cited by 138 publications

(71 citation statements)

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“…3 D and E). During the reconstitution of apo-Mb with heme, two binding modes of the heme are observed at an early stage (25). Even for the heme⅐HO complex, a minor conformation has been reported in solution that is influenced by interactions of the two heme propionic acid groups (26).…”

Section: Resultsmentioning

confidence: 99%

Design of metal cofactors activated by a protein–protein electron transfer system

Ueno¹,

Yokoi

Unno

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Protein-to-protein electron transfer (ET) is a critical process in biological chemistry for which fundamental understanding is expected to provide a wealth of applications in biotechnology. Investigations of protein-protein ET systems in reductive activation of artificial cofactors introduced into proteins remains particularly challenging because of the complexity of interactions between the cofactor and the system contributing to ET. In this work, we construct an artificial protein-protein ET system, using heme oxygenase (HO), which is known to catalyze the conversion of heme to biliverdin. HO uses electrons provided from NADPH/ cytochrome P450 reductase (CPR) through protein-protein complex formation during the enzymatic reaction. We report that a Fe III (Schiff-base), in the place of the active-site heme prosthetic group of HO, can be reduced by NADPH/CPR. The crystal structure of the Fe(10-CH2CH2COOH-Schiff-base)⅐HO composite indicates the presence of a hydrogen bond between the propionic acid carboxyl group and Arg-177 of HO. Furthermore, the ET rate from NADPH/ CPR to the composite is 3.5-fold faster than that of Fe(Schiffbase)⅐HO, although the redox potential of Fe(10-CH2CH2COOH-Schiff-base)⅐HO (؊79 mV vs. NHE) is lower than that of Fe(Schiffbase)⅐HO (؉15 mV vs. NHE), where NHE is normal hydrogen electrode. This work describes a synthetic metal complex activated by means of a protein-protein ET system, which has not previously been reported. Moreover, the result suggests the importance of the hydrogen bond for the ET reaction of HO. Our Fe(Schiffbase)⅐HO composite model system may provide insights with regard to design of ET biosystems for sensors, catalysts, and electronics devices.heme oxygenase ͉ metalloprotein ͉ protein-protein interaction ͉ schiff-base ͉ hydrogen bond D esign of biological protein-protein electron transfer (ET) systems (1-6) for the creation of artificial catalysis and bioelectronics devices for novel biosensors is a challenging objective (7-9). Several researchers have made significant advances in construction of artificial ET biosystems by chemical modification of native cofactors (10-12), substrates (13), and enzymes (14). For example, Gray and Winkler (6) reported that synthetic light harvesting metal complexes bound to metalloproteins can act as triggers of ET reactions. However, challenges remain for introduction of unnatural molecules into protein scaffolds in design of ET systems because of the challenges in understanding the complex noncovalent interactions contributing to ET processes.Heme oxygenase (HO) is known to catalyze the conversion of heme to biliverdin. HO utilizes electrons provided from a NADPH͞cytochrome P450 reductase (CPR) system (Fig. 1A) (15). The first electron reduces the heme Fe(III) to Fe(II) to initiate the enzymatic reaction, which is followed by a second reduction of O 2 -ligated heme for the hydroxylation of the C ␣ atom of the heme (Fig. 1 A). The electron flow has been proposed to proceed from NADPH to the heme iron atom of the redox partner, HO, v...

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Section: Resultsmentioning

confidence: 99%

Design of metal cofactors activated by a protein–protein electron transfer system

Ueno¹,

Yokoi

Unno

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Addition of denaturants such as acid, base or organic cosolvents can induce protein unfolding and disruption of the native heme-protein interactions, thus generating apo-myoglobin (aMb) and free heme. The solution-phase transitions from hMb to aMb and vice versa have been extensively studied, both optically [43][44][45] and by ESI-MS [29, 39, 46 -51]. The results of this work demonstrate that diffusion measurements by ESI-MS are a sensitive method for probing noncovalent heme-protein interactions in solution.…”

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confidence: 79%

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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“…It was not possible to evaluate this ratio directly in the absence of exogenous ligand, but cyanide binding to the His46 variants is faster than to wild-type and reorientation in the cyanomet form is expected to be slow [61].…”

Section: Discussionmentioning

confidence: 99%

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

Nothnagel

Love

Lecomte

2009

Journal of Inorganic Biochemistry

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Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: evidence for intramolecular reorientation about a heme two-fold axis

Cited by 138 publications

References 1 publication

Design of metal cofactors activated by a protein–protein electron transfer system

Design of metal cofactors activated by a protein–protein electron transfer system

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

The role of the heme distal ligand in the post-translational modification of Synechocystis hemoglobin

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