ESR of Iron Proteins

Smith, Thomas D.; Pilbrow, John R.

doi:10.1007/978-1-4615-6537-6_3

Cited by 22 publications

(10 citation statements)

References 508 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The EPR spectrum of ferric sGC reported here displays rhombic symmetry (Figure 1). The g values obtained from the simulation of the spectrum are indicative of high-spin iron (Cammack & Cooper, 1993;Smith & Pilbrow, 1980). The EPR spectra of 6-coordinate high-spin heme complexes, such as metHb, tend to display axial symmetry, with a single feature near g ) 6, whereas the EPR spectra of 5-coordinate high-spin heme complexes usually show a rhombic distortion with two features near g ) 6 (Goodin & McRee, 1993).…”

Section: Discussionmentioning

confidence: 99%

Spectral and Ligand-Binding Properties of an Unusual Hemoprotein, the Ferric Form of Soluble Guanylate Cyclase

et al. 1996

View full text Add to dashboard Cite

The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (.NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. .NO increases the Vmax of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme has been obtained. The spectrum displays rhombic symmetry and is indicative of a high-spin heme. Computer simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0 with linewidths of 3.3, 4.1, and 3.3 mT, respectively. Using electronic absorption spectroscopy, it was observed that the ferric heme binds cyanide to form a 6-coordinate low-spin complex. The rate constants for association (k(on)) and dissociation (k(off)) of cyanide at 10 degrees C have been determined to be (7.8 +/- 0.3) x 10(-2) M(-1) s(- 1) and (7.2 +/- 0.2) x 10(-5) s(-1), respectively. Unlike the ferrous form of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a Kd of 26 +/- 4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of approximately 57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon the oxidation of the heme iron.

show abstract

Section: Discussionmentioning

confidence: 99%

Spectral and Ligand-Binding Properties of an Unusual Hemoprotein, the Ferric Form of Soluble Guanylate Cyclase

et al. 1996

View full text Add to dashboard Cite

show abstract

“…Electron paramagnetic resonance (EPR) spectra ofH175G and wild-type CCP are compared in Fig. 3a. As isolated, the wild-type enzyme exhibits a high-spin ferric EPR signal with a small rhombic distortion (g. = 6.56, gy = 5.20) that is critically dependent on the geometry of axial ligand coordination (9,20). The EPR spectrum of bisaquo H175G also reflects a high-spin ferric state but indicates that the protein is a mixture of axial (gL = 5.99) and rhombic (g,, = 6.59, gy = 5.37) components (Fig.…”

Section: Methodsmentioning

confidence: 99%

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

McRee

Jensen

Fitzgerald

et al. 1994

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

View full text Add to dashboard Cite

The crystal structure of the His-175 -) Gly (H175G) mutant of cytochrome-c peroxidase (EC 1.11.1.5), missing its only heme ligand, reveals that the histidine is replaced by solvent to give a bisaquo heme protein. This protein retains some residual activity, which can be stimulated or inhibited by addition of exogenous ligands. Structural analysis confirms the binding of imidazole to the heme at the position of the wild-type histidine ligand. This imidazole complex reacts readily with hydrogen peroxide to produce a radical species with novel properties. However, reactivation in this complex is incomplete (""5%), which, in view ofthe very similar structures of the wild-type and the H175G/imidazole forms, implies a critical role for tethering of the axial ligand in catalysis. This study demonstrates the feasibility of constructing heme enzymes with no covalent link to the protein and with unnatural ligand replacements. Such enzymes may prove useful in studies of electron transfer mechanisms and in the engineering of novel heme-based catalysts.The parameters required of the axial ligands for defining the diverse chemistry of heme enzymes are elusive. Peroxidases often contain a histidine ligand to the iron, whereas cysteine is found in monooxygenases and tyrosine in catalases (1, 2). Cytochrome-c peroxidase (CCP; EC 1.11.1.5) catalyzes the oxidation of cytochrome c (cyt c) by H202 through an intermediate state (ES) that consists of a ferryl (Fe4+=O) heme and a free radical localized on Trp-191 (3). The buried carboxylate of Asp-235 stabilizes this free radical and also accepts a hydrogen bond from the histidine heme ligand, His-175. The resulting Asp/His/metal triad is a common motif in metalloproteins and is reminiscent of the Asp/His/ Ser triad of serine proteases (4,5). In this study, we report the deletion of the histidine iron ligand of CCP to produce a heme enzyme which contains no covalent link to the protein. The facile occupation ofthis cavity by exogenous small molecules demonstrates the potential for producing heme catalysts containing a wide range of unnatural ligands. MATERIALS AND METHODSProtein Expression and Purification. Wild-type and mutant CCP proteins were overexpressed in Escherichia coli BL21(DE3) using the plasmid pT7CCP (6). Wild-type CCP in our laboratory is that with Met-Lys-Thr at the N terminus and containing Ile-53 and Gly-152 (7). The His-175 --Gly (H175G) mutant was created by site-directed mutagenesis, expressed, purified to homogeneity, and reconstituted with heme as described (6). This purified reconstituted protein was crystallized twice against distilled water and stored as a crystal suspension at 77 K. Protein concentrations were determined from the extinction coefficients determined by pyridine hemochromogens (8, 9). In the case of H175G the extinction coefficient was determined for the 280-nm band because of the variability (with pH and temperature) in the intensity and energy of the Soret band (see below).X-Ray Crystallography.t Attempts to obtain crystals of H175G by ...

show abstract

“…37 The HS resonances contributed a factor of about 3.5 over the total protein concentration, equivalent to 0.9 Fe/monomer. The feature observed at g = 4.3 was assigned to iron impurities and not considered in this study.…”

mentioning

confidence: 99%

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

et al. 2011

View full text Add to dashboard Cite

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans. It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = 5/2 high-spin (HS) and Fe(III) S = 1/2 low-spin (LS) hemes. 14N ENDOR spectroscopy on the S = 5/2 signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = 5/2 heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T 1-spin–lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

show abstract

ESR of Iron Proteins

Cited by 22 publications

References 508 publications

Spectral and Ligand-Binding Properties of an Unusual Hemoprotein, the Ferric Form of Soluble Guanylate Cyclase

Spectral and Ligand-Binding Properties of an Unusual Hemoprotein, the Ferric Form of Soluble Guanylate Cyclase

Construction of a bisaquo heme enzyme and binding by exogenous ligands.

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

Contact Info

Product

Resources

About