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James E. Erman - One of the best experts on this subject based on the ideXlab platform.
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CharaCterization of a Covalently linked yeast CytoChrome C-CytoChrome C Peroxidase Complex: evidenCe for a single, CatalytiCally aCtive CytoChrome C binding site on CytoChrome C Peroxidase.
Biochemistry, 2006Co-Authors: Siddartha Nakani, Thanarat Viriyakul, Robert A. Mitchell, Lidia B. Vitello, James E. ErmanAbstract:A Covalent Complex between reCombinant yeast iso-1-CytoChrome C and reCombinant yeast CytoChrome C Peroxidase (rCCP), in whiCh the CrystallographiCally defined CytoChrome C binding site [Pelletier, H., and Kraut, J. (1992) SCienCe 258, 1748−1755] is bloCked, was synthesized via disulfide bond formation using speCifiCally engineered Cysteine residues in both yeast iso-1-CytoChrome C and yeast CytoChrome C Peroxidase [Papa, H. S., and Poulos, T. L. (1995) BioChemistry 34, 6573−6580]. Previous studies on similar Covalent Complexes, those that bloCk the Pelletier−Kraut CrystallographiC site, have demonstrated that samples of the Covalent Complexes have deteCtable aCtivities that are signifiCantly lower than those of wild-type yCCP, usually in the range of ∼1−7% of that of the wild-type enzyme. Using gradient elution proCedures in the purifiCation of the engineered Peroxidase, CytoChrome C, and Covalent Complex, along with aCtivity measurements during the purifiCation steps, we demonstrate that the residual aC...
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pH DependenCe of heme iron Coordination, hydrogen peroxide reaCtivity, and Cyanide binding in CytoChrome C Peroxidase(H52K).
Biochemistry, 2004Co-Authors: Miriam C. Foshay, Lidia B. Vitello, James E. ErmanAbstract:ReplaCement of the distal histidine, His-52, in CytoChrome C Peroxidase (CCP) with a lysine residue produCes a mutant CytoChrome C Peroxidase, CCP(H52K), with speCtral and kinetiC properties signif...
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yeast CytoChrome C Peroxidase meChanistiC studies via protein engineering
Biochimica et Biophysica Acta, 2002Co-Authors: James E. Erman, Lidia B. VitelloAbstract:CytoChrome C Peroxidase (CCP) is a yeast mitoChondrial enzyme that Catalyzes the reduCtion of hydrogen peroxide to water by ferroCytoChrome C. It was the first heme enzyme to have its CrystallographiC struCture determined and, as a ConsequenCe, has played a pivotal role in developing ideas about struCtural Control of heme protein reaCtivity. GenetiC engineering of the aCtive site of CCP, along with struCtural, speCtrosCopiC, and kinetiC CharaCterization of the mutant proteins has provided Considerable insight into the meChanism of hydrogen peroxide aCtivation, oxygen-oxygen bond Cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The CatalytiC meChanism involves Complex formation between CytoChrome C and CCP. The CytoChrome C/CCP system has been very useful in eluCidating the Complexities of long-range eleCtron transfer in biologiCal systems, inCluding protein-protein reCognition, Complex formation, and intraComplex eleCtron transfer proCesses.
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CytoChrome C/CytoChrome C Peroxidase Complex: EffeCt of Binding-Site Mutations on the ThermodynamiCs of Complex Formation†
Biochemistry, 1997Co-Authors: James E. Erman, Lidia B. Vitello, Gordon C. Kresheck, Mark A. MillerAbstract:The CytoChrome C/CytoChrome C Peroxidase system has been extensively investigated as a model for long-range eleCtron transfer in biology. Two models for the struCture of the one-to-one CytoChrome C/CytoChrome C Peroxidase Complex in solution exist: one is based upon Computer doCking of the two proteins and the seCond is based upon the struCture of the Complex in the Crystalline state. Titration Calorimetry is used to investigate the interaCtion of horse ferriCytoChrome C with baker's yeast CytoChrome C Peroxidase and with six CytoChrome C Peroxidase mutants. Five of the six Peroxidase mutants eliminate a negative Charge in the CytoChrome C binding site by replaCing a side-Chain Carboxylate with an amide. The sixth mutation replaCes a surfaCe alanine residue with phenylalanine. The binding affinity between CytoChrome C and the CytoChrome C Peroxidase mutants varies from no signifiCant Change in Comparison to the wild-type enzyme to a 4-fold deCrease in the equilibrium assoCiation Constant. The pattern of deCreasing CytoChrome C binding affinity for the CytoChrome C Peroxidase mutants is Consistent with the CytoChrome C binding domain defined by X-ray Crystallography [Pelletier, H., & Kraut, J. (1992) SCienCe 258, 1748-1755]. For those mutants whiCh have lower affinity for CytoChrome C, the lower affinity is due to a deCrease in the entropy Change upon Complex formation, Consistent with the differenCe in hydration of Carboxylate and amide groups.
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Oxidation of yeast iso-1 ferroCytoChrome C by yeast CytoChrome C Peroxidase Compounds I and II. DependenCe upon ioniC strength.
Biochemistry, 1995Co-Authors: Andrea L. Matthis, Lidia B. Vitello, James E. ErmanAbstract:The reduCtion of CytoChrome C Peroxidase Compound I by exCess yeast iso-1 ferroCytoChrome C is biphasiC. Two pseudo-first-order rate Constants Can be measured by stopped-flow teChniques. The fastest rate proCess is the reduCtion of CytoChrome C Peroxidase Compound I to Compound II, and the slower proCess is the reduCtion of II to the native enzyme. The yeast iso-1 ferroCytoChrome C ConCentration dependenCe of the reduCtion of CytoChrome C Peroxidase Compound I to Compound II is Consistent with a meChanism involving two binding sites for CytoChrome C on CytoChrome C Peroxidase. EleCtron transfer from CytoChrome C bound at the high-affinity binding site to the Fe(IV) site in CytoChrome C Peroxidase Compound I is dependent upon ioniC strength, inCreasing from 15 +/- 6 to 2000 +/- 100 s-1 over the ioniC strength range 0.01-0.20 M. The reduCtion rate of the Fe(IV) site in the 2:1 yeast iso-1 ferroCytoChrome C/CytoChrome C Peroxidase Compound I Complex is essentially independent of ioniC strength with a value of 3800 +/- 300 s-1. The Fe(IV) site in CytoChrome C Peroxidase Compound I is preferentially reduCed by yeast ferroCytoChrome C between 0.01 and 0.20 M ioniC strength while the Trp-191 radiCal is preferentially reduCed above 0.30 M ioniC strength. The assoCiation rate Constant for the binding of yeast iso-1 ferroCytoChrome C to CytoChrome C Peroxidase Compound I Can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ioniC strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ioniC strength. Between 0.01 and 0.20 M ioniC strength, the reduCtion of CytoChrome C Peroxidase Compound II to the native enzyme is anomalous. The reaCtion is independent of the CytoChrome C ConCentration and direCtly proportional to the initial CytoChrome C Peroxidase Compound I ConCentration.
Lidia B. Vitello - One of the best experts on this subject based on the ideXlab platform.
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CharaCterization of a Covalently linked yeast CytoChrome C-CytoChrome C Peroxidase Complex: evidenCe for a single, CatalytiCally aCtive CytoChrome C binding site on CytoChrome C Peroxidase.
Biochemistry, 2006Co-Authors: Siddartha Nakani, Thanarat Viriyakul, Robert A. Mitchell, Lidia B. Vitello, James E. ErmanAbstract:A Covalent Complex between reCombinant yeast iso-1-CytoChrome C and reCombinant yeast CytoChrome C Peroxidase (rCCP), in whiCh the CrystallographiCally defined CytoChrome C binding site [Pelletier, H., and Kraut, J. (1992) SCienCe 258, 1748−1755] is bloCked, was synthesized via disulfide bond formation using speCifiCally engineered Cysteine residues in both yeast iso-1-CytoChrome C and yeast CytoChrome C Peroxidase [Papa, H. S., and Poulos, T. L. (1995) BioChemistry 34, 6573−6580]. Previous studies on similar Covalent Complexes, those that bloCk the Pelletier−Kraut CrystallographiC site, have demonstrated that samples of the Covalent Complexes have deteCtable aCtivities that are signifiCantly lower than those of wild-type yCCP, usually in the range of ∼1−7% of that of the wild-type enzyme. Using gradient elution proCedures in the purifiCation of the engineered Peroxidase, CytoChrome C, and Covalent Complex, along with aCtivity measurements during the purifiCation steps, we demonstrate that the residual aC...
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pH DependenCe of heme iron Coordination, hydrogen peroxide reaCtivity, and Cyanide binding in CytoChrome C Peroxidase(H52K).
Biochemistry, 2004Co-Authors: Miriam C. Foshay, Lidia B. Vitello, James E. ErmanAbstract:ReplaCement of the distal histidine, His-52, in CytoChrome C Peroxidase (CCP) with a lysine residue produCes a mutant CytoChrome C Peroxidase, CCP(H52K), with speCtral and kinetiC properties signif...
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yeast CytoChrome C Peroxidase meChanistiC studies via protein engineering
Biochimica et Biophysica Acta, 2002Co-Authors: James E. Erman, Lidia B. VitelloAbstract:CytoChrome C Peroxidase (CCP) is a yeast mitoChondrial enzyme that Catalyzes the reduCtion of hydrogen peroxide to water by ferroCytoChrome C. It was the first heme enzyme to have its CrystallographiC struCture determined and, as a ConsequenCe, has played a pivotal role in developing ideas about struCtural Control of heme protein reaCtivity. GenetiC engineering of the aCtive site of CCP, along with struCtural, speCtrosCopiC, and kinetiC CharaCterization of the mutant proteins has provided Considerable insight into the meChanism of hydrogen peroxide aCtivation, oxygen-oxygen bond Cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The CatalytiC meChanism involves Complex formation between CytoChrome C and CCP. The CytoChrome C/CCP system has been very useful in eluCidating the Complexities of long-range eleCtron transfer in biologiCal systems, inCluding protein-protein reCognition, Complex formation, and intraComplex eleCtron transfer proCesses.
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CytoChrome C/CytoChrome C Peroxidase Complex: EffeCt of Binding-Site Mutations on the ThermodynamiCs of Complex Formation†
Biochemistry, 1997Co-Authors: James E. Erman, Lidia B. Vitello, Gordon C. Kresheck, Mark A. MillerAbstract:The CytoChrome C/CytoChrome C Peroxidase system has been extensively investigated as a model for long-range eleCtron transfer in biology. Two models for the struCture of the one-to-one CytoChrome C/CytoChrome C Peroxidase Complex in solution exist: one is based upon Computer doCking of the two proteins and the seCond is based upon the struCture of the Complex in the Crystalline state. Titration Calorimetry is used to investigate the interaCtion of horse ferriCytoChrome C with baker's yeast CytoChrome C Peroxidase and with six CytoChrome C Peroxidase mutants. Five of the six Peroxidase mutants eliminate a negative Charge in the CytoChrome C binding site by replaCing a side-Chain Carboxylate with an amide. The sixth mutation replaCes a surfaCe alanine residue with phenylalanine. The binding affinity between CytoChrome C and the CytoChrome C Peroxidase mutants varies from no signifiCant Change in Comparison to the wild-type enzyme to a 4-fold deCrease in the equilibrium assoCiation Constant. The pattern of deCreasing CytoChrome C binding affinity for the CytoChrome C Peroxidase mutants is Consistent with the CytoChrome C binding domain defined by X-ray Crystallography [Pelletier, H., & Kraut, J. (1992) SCienCe 258, 1748-1755]. For those mutants whiCh have lower affinity for CytoChrome C, the lower affinity is due to a deCrease in the entropy Change upon Complex formation, Consistent with the differenCe in hydration of Carboxylate and amide groups.
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Oxidation of yeast iso-1 ferroCytoChrome C by yeast CytoChrome C Peroxidase Compounds I and II. DependenCe upon ioniC strength.
Biochemistry, 1995Co-Authors: Andrea L. Matthis, Lidia B. Vitello, James E. ErmanAbstract:The reduCtion of CytoChrome C Peroxidase Compound I by exCess yeast iso-1 ferroCytoChrome C is biphasiC. Two pseudo-first-order rate Constants Can be measured by stopped-flow teChniques. The fastest rate proCess is the reduCtion of CytoChrome C Peroxidase Compound I to Compound II, and the slower proCess is the reduCtion of II to the native enzyme. The yeast iso-1 ferroCytoChrome C ConCentration dependenCe of the reduCtion of CytoChrome C Peroxidase Compound I to Compound II is Consistent with a meChanism involving two binding sites for CytoChrome C on CytoChrome C Peroxidase. EleCtron transfer from CytoChrome C bound at the high-affinity binding site to the Fe(IV) site in CytoChrome C Peroxidase Compound I is dependent upon ioniC strength, inCreasing from 15 +/- 6 to 2000 +/- 100 s-1 over the ioniC strength range 0.01-0.20 M. The reduCtion rate of the Fe(IV) site in the 2:1 yeast iso-1 ferroCytoChrome C/CytoChrome C Peroxidase Compound I Complex is essentially independent of ioniC strength with a value of 3800 +/- 300 s-1. The Fe(IV) site in CytoChrome C Peroxidase Compound I is preferentially reduCed by yeast ferroCytoChrome C between 0.01 and 0.20 M ioniC strength while the Trp-191 radiCal is preferentially reduCed above 0.30 M ioniC strength. The assoCiation rate Constant for the binding of yeast iso-1 ferroCytoChrome C to CytoChrome C Peroxidase Compound I Can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ioniC strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ioniC strength. Between 0.01 and 0.20 M ioniC strength, the reduCtion of CytoChrome C Peroxidase Compound II to the native enzyme is anomalous. The reaCtion is independent of the CytoChrome C ConCentration and direCtly proportional to the initial CytoChrome C Peroxidase Compound I ConCentration.
Thomas L. Poulos - One of the best experts on this subject based on the ideXlab platform.
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leishmania major Peroxidase is a CytoChrome C Peroxidase
Biochemistry, 2012Co-Authors: Victoria S Jasion, Thomas L. PoulosAbstract:Leishmania major Peroxidase (LmP) exhibits both asCorbate and CytoChrome C Peroxidase aCtivities. Our previous results illustrated that LmP has a muCh higher aCtivity against horse heart CytoChrome C than asCorbate, suggesting that CytoChrome C may be the biologiCally important substrate. To eluCidate the biologiCal funCtion of LmP, we have reCombinantly expressed, purified, and determined the 2.08 A Crystal struCture of L. major CytoChrome C (LmCytC). Like other types of CytoChrome C, LmCytC has an eleCtropositive surfaCe surrounding the exposed heme edge that serves as the site of doCking with redox partners. KinetiC assays performed with LmCytC and LmP show that LmCytC is a muCh better substrate for LmP than horse heart CytoChrome C. Furthermore, unlike the well-studied yeast system, the reaCtion follows ClassiC MiChaelis–Menten kinetiCs and is sensitive to an inCreasing ioniC strength. Using the yeast CoCrystal as a Control, protein–protein doCking was performed using Rosetta to develop a model for th...
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engineering asCorbate Peroxidase aCtivity into CytoChrome C Peroxidase
Biochemistry, 2008Co-Authors: Yergalem T Meharenna, B. Bhaskar, Patricia Oertel, Thomas L. PoulosAbstract:CytoChrome C Peroxidase (CCP) and asCorbate Peroxidase (APX) have very similar struCtures, and yet neither CCP nor APX exhibit eaCh others aCtivities with respeCt to reduCing substrates. APX has a unique substrate binding site near the heme propionates where asCorbate H-bonds with a surfaCe Arg and one heme propionate (Sharp et al. (2003) Nat. StruC. Biol. 10, 303–307). The Corresponding region in CCP has a muCh longer surfaCe loop and the CritiCal Arg residue that is required for asCorbate binding in APX is Asn in CCP. In order to Convert CCP into an APX, the asCorbate binding loop and CritiCal arginine were engineered into CCP to give the CCP2APX mutant. The mutant Crystal struCture shows that the engineered site is nearly identiCal to that found in APX. While wild type CCP shows no APX aCtivity, CCP2APX Catalyzes the peroxidation of asCorbate at a rate of ≈ 12 min−1 indiCating that the engineered asCorbate binding loop Can bind asCorbate.
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The 1.13-A struCture of iron-free CytoChrome C Peroxidase.
JBIC Journal of Biological Inorganic Chemistry, 2005Co-Authors: B. Bhaskar, Thomas L. PoulosAbstract:The iron-free CytoChrome C Peroxidase (CCP) Crystal struCture has been determined to 1.13 A and Compared with the 1.2-A ferriC-CCP struCture. Quite unexpeCtedly, removal of the iron has no effeCt on porphyrin geometry and distortion, indiCating that protein–porphyrin interaCtions and not iron Coordination or formation of the axial His–Fe bond determines porphyrin Conformation. However, there are Changes in solvent struCture in the distal poCket, whiCh lead to Changes in the distal His52 aCid–base Catalyst. The observed ability of His52 to move in response to small Changes in solvent struCture is very likely important for its role as a Catalyst in assisting in the heterolytiC fission of the peroxide O–O bond.
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Mesopone CytoChrome C Peroxidase: funCtional model of heme oxygenated oxidases.
Journal of Inorganic Biochemistry, 2002Co-Authors: Chad E. Immoos, B. Bhaskar, Michael S. Cohen, Tiffany P. Barrows, Patrick J. Farmer, Thomas L. PoulosAbstract:AbstraCt The effeCt of heme ring oxygenation on enzyme struCture and funCtion has been examined in a reConstituted CytoChrome C Peroxidase. OxoChlorin derivatives were formed by OsO 4 treatment of mesoporphyrin followed by aCid-Catalyzed pinaCol rearrangement. The northern oxoChlorin isomers were isolated by Chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1 H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl 2 and reConstituted into CytoChrome C Peroxidase (CCP) forming a hybrid green protein, MpCCP. The heme-altered enzyme has 99% wild-type Peroxidase aCtivity with CytoChrome C . EPR speCtrosCopy of MpCCP intermediate Compound I verifies the formation of the Trp 191 radiCal similar to wild-type CCP in the reaCtion CyCle. Peroxidase aCtivity with small moleCules is varied: guaiaCol turnover inCreases approximately five-fold while that with ferroCyanide is ∼85% of native. The eleCtron-withdrawing oxo-substitutents on the CofaCtor Cause a ∼60-mV inCrease in Fe III /Fe II reduCtion potential. The present investigation represents the first struCtural CharaCterization of an oxoChlorin protein with X-ray intensity data ColleCted to 1.70 A. Although a mixture of R - and S -mesopone isomers of the FeMP CofaCtor was used during heme inCorporation into the apo-protein, only the S -isomer is found in the Crystallized protein.
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Cation-induCed stabilization of the engineered Cation-binding loop in CytoChrome C Peroxidase (CCP).
Biochemistry, 2002Co-Authors: B. Bhaskar, Christopher A. Bonagura, Thomas L. PoulosAbstract:We have previously shown that the K+ site found in the proximal heme poCket of asCorbate Peroxidase (APX) Could be suCCessfully engineered into the Closely homologous CytoChrome C Peroxidase (CCP) ...
Marcellus Ubbink - One of the best experts on this subject based on the ideXlab platform.
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interaCtion of yeast iso 1 CytoChrome C with CytoChrome C Peroxidase investigated by 15n 1h heteronuClear nmr speCtrosCopy
Biochemistry, 2001Co-Authors: Jonathan A R Worrall, Urszula Kolczak, Gerard W Canters, Marcellus UbbinkAbstract:The interaCtion of yeast iso-1-CytoChrome C with its physiologiCal redox partner CytoChrome C Peroxidase has been investigated using heteronuClear NMR teChniques. ChemiCal shift perturbations for both 15N and 1H nuClei arising from the interaCtion of isotopiCally enriChed 15N CytoChrome C with CytoChrome C Peroxidase have been observed. For the diamagnetiC, ferrous CytoChrome C, 34 amides are affeCted by binding, Corresponding to residues at the front faCe of the protein and in agreement with the interfaCe observed in the 1:1 Crystal struCture of the Complex. In Contrast, for the paramagnetiC, ferriC protein, 56 amides are affeCted, Corresponding to residues both at the front and toward the rear of the protein. In addition, the ChemiCal shift perturbations were larger for the ferriC protein. Using experimentally observed pseudoContaCt shifts the magnetiC susCeptibility tensor of yeast iso-1-CytoChrome C in both the free and bound forms has been CalCulated with HN nuClei as inputs. In Contrast to an earlie...
James D. Satterlee - One of the best experts on this subject based on the ideXlab platform.
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CytoChrome C Peroxidase Complexed with CytoChrome C has an unperturbed heme moiety.
Biochemistry, 1996Co-Authors: Jianling Wang, James D. Satterlee, Susan J. Moench, Randy W. Larsen, Denis L. Rousseau, Mark R. OndriasAbstract:Transient resonanCe Raman, Raman differenCe, CirCular diChroism (CD), and optiCal absorption studies have been Carried out on the eleCtrostatiC Complexes formed by yeast CytoChrome C Peroxidase (CC...
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The effeCt of the Asn82-->Asp mutation in yeast CytoChrome C Peroxidase studied by proton NMR speCtrosCopy.
European Journal of Biochemistry, 1994Co-Authors: James D. Satterlee, James E. Erman, Steve L. Alam, J. Matthew Mauro, Thomas L. PoulosAbstract:Proton NMR studies of the mutant of baker's yeast CytoChrome C Peroxidase-Cyanide with the Asn 82Asp mutation ([N82D]CytoChrome C Peroxidase-CN) are presented and Compared to the wild-type enzyme. This mutation alters an amino aCid that forms a hydrogen bond to His52, the distal histidine residue that interaCts in the heme poCket with heme-bound ligands. His52 is an important partiCipant in the initial hydrogen peroxide deComposition step of CytoChrome C Peroxidase. In wild-type CytoChrome C Peroxidase-CN, His52 hydrogen bonds to the neighboring Am82 peptide Carbonyl group and to heme-Coordinated Cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The prinCipal result from this study is the observation that three hyperfine-shifted resonanCes disappear from the speCtrum of [N82D] CytoChrome C Peroxidase-CN Compared to the wild-type enzyme. All three absent resonanCes in [N82D]CytoChrome C Peroxidase-CN belong to His52 and this leads to the ConClusion that the result of the mutation has been elimination of the His52–Am82 and His52–heme-Coordinated Cyanide hydrogen bonds.
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Proton NMR studies of nonCovalent Complexes of CytoChrome C Peroxidase-Cyanide with horse and yeast ferriCytoChromes C
Biochemistry, 1993Co-Authors: James E. Erman, James D. SatterleeAbstract:NonCovalent Complexes of Cyanide-ligated CytoChrome C Peroxidase with horse ferriCytoChrome C and yeast isozyme-1 ferriCytoChrome C have been formed in 10 mM potassium nitrate salt solutions and studied by proton NMR speCtrosCopy. The ChemiCal shifts in the ferriCytoChrome C speCtrum induCed by Complex formation with low-spin, Cyanide-ligated CytoChrome C Peroxidase are similar to the Corresponding shifts induCed by Complex formation with resting-state CytoChrome C Peroxidase, found previously. As with the resting-state enzyme, the Complex between yeast CytoChrome C and CytoChrome C Peroxidase-Cyanide exhibits the larger set of Complex-induCed shifts. Two-dimensional proton NMR speCtrosCopy has been used to make resonanCe assignments. This was neCessitated due to the extensive resonanCe overlap between the two proteins in the hyperfine shift region, sinCe both heme proteins in this Complex are low-spin paramagnetiC speCies. These results expand preliminary work that revealed for the first time that CytoChrome C binding affeCted the resonanCes of protons in the Peroxidase heme poCket [Yi, Q., Erman, J. E., & Satterlee, J. D. (1992) J. Am. Chem. SoC. 114, 7907-7909]. The pattern of CytoChrome C Peroxidase Complex-induCed shifts is largely Consistent with the X-ray Crystal struCtures of these two Complexes that have reCently been published.
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Proton NMR Comparison of nonCovalent and Covalently Cross-linked Complexes of CytoChrome C Peroxidase with horse, tuna, and yeast ferriCytoChromes C.
Biochemistry, 1992Co-Authors: Susan J. Moench, James E. Erman, Stamatia Chroni, Bih-show Lou, James D. SatterleeAbstract:Proton NMR speCtrosCopy at 500 and 361 MHz has been used to CharaCterize the nonCovalent or eleCtrostatiC Complexes of yeast CytoChrome C Peroxidase (CCP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferriCytoChromes C and the Covalently Cross-linked Complexes of CytoChrome C Peroxidase with horse and yeast isozyme-1 ferriCytoChromes C. Under the Conditions employed in this work, the stoiChiometry of the predominant Complex formed in solution (whiCh totaled greater than 90% of Complex formed) was found to be 1:1 in all Cases. These studies have eluCidated signifiCant differenCes in the proton NMR absorption speCtra and the one-dimensional nuClear Overhauser effeCt differenCe speCtra of the Complexes, depending on the speCifiC speCies of ferriCytoChrome C inCorporated. In partiCular, the results indiCate that the nonCovalent Complexes formed between CCP and physiologiCal redox partners (yeast isozyme-1 or yeast isozyme-2 ferriCytoChromes C) are distinCtly different from the nonCovalent Complexes formed between CCP and ferriCytoChromes C from horse and tuna. Parallel ChemiCal Cross-linking studies Carried out using mixtures of CytoChrome C Peroxidase with horse ferriCytoChrome C, and CytoChrome C Peroxidase with yeast isozyme-1 ferriCytoChrome C further emphasize suCh CytoChrome C-dependent differenCes, with only the Covalently Cross-linked Complex of physiologiCal redox partners (CytoChrome C Peroxidase/yeast isozyme-1) displaying NMR speCtra CharaCteristiC of a heterogeneous mixture of different 1:1 Complexes. Finally, one-dimensional nuClear Overhauser effeCt experiments have proven valuable in seleCtively and effiCiently probing the protein-protein interfaCe in these Complexes, inCluding the environment around the CytoChrome C heme 3-methyl group and Phe-82.
