Role of the Heme Propionates in the Interaction of Heme with Apomyoglobin and Apocytochrome b 5

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...

Section: Resultsmentioning

confidence: 99%

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

Ueno¹,

Yokoi

Unno

et al. 2006

“…The small decrease in thermal stability observed for the variants can be understood in terms of the loss of the hydrogen bond to the heme propionate upon replacement of K45 and a small destabilizing electrostatic interaction between the heme propionate group and E63. The role of hydrogen bonding interactions between the heme propionates and surface residues of Mb has been studied more extensively in previous studies (39,40).…”

Section: Discussionmentioning

confidence: 99%

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Hunter

Maurus²,

Mauk³

et al. 2003

Self Cite

A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional 1 H NMR spectroscopy indicates that Mn 2؉ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn 2؉ is 1.28(4) ؋ 10 4 M ؊1 (pH 6.96, ionic strength I ‫؍‬ 17.2 M, 25°C). In addition, these substitutions lower the reduction potential of the protein and increase the pK a for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn 2؉ -peroxidase activity of the variant are both increased Ϸ3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn 2؉ . The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn 2؉ and Cd 2؉ . Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.

“…The 2.4°C decrease in T m for the quadruple variant presumably results from elimination of the hydrogen bond between the heme propionate and K45 in this protein. Elimination of this hydrogen bond destabilizes the binding of heme to apomyoglobin (47), and decreased stability of the heme-apomyoglobin complex is known to be correlated with decreased thermal stability of the holoprotein (48).…”

Section: Discussionmentioning

confidence: 99%

In vitro evolution of horse heart myoglobin to increase peroxidase activity

Wan

Twitchett

Eltis

et al. 1998

Self Cite

104

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay.