1.11.1.5 A124K/K128A site-directed mutagenesis, no significant changes 705165 1.11.1.5 A193F surface mutant, shift in reduction potential to -170 mV. Analysis of spectroscopic properties 675241 1.11.1.5 A193W mutant designed to incorporate a Trp-based extension to move oxidizing equivalents from the heme to the protein surface. Mutant is able to oxidize veratryl alcohol substrate with turnover numbers greater than wild type 742339 1.11.1.5 A193W/Y229W mutant designed to incorporate a Trp-based extension to move oxidizing equivalents from the heme to the protein surface. Mutant is able to oxidize veratryl alcohol substrate with turnover numbers greater than wild type, possibly using an electron hopping mechanism 742339 1.11.1.5 D146N surface mutant, shift in reduction potential to -173 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D146N/D148N surface mutant, shift in reduction potential to -173 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D18K positive-to-negative charge-reversal mutant 685179 1.11.1.5 D210K positive-to-negative charge-reversal mutant 685179 1.11.1.5 D235A proximal pocket mutant, shift in reduction potential to -78 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D235E proximal pocket mutant, shift in reduction potential to -113 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D235N predominantly hexacoordinate between pH 4 and pH8 394630 1.11.1.5 D235N proximal pocket mutant, shift in reduction potential to -79 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D33K positive-to-negative charge-reversal mutant 685179 1.11.1.5 D34K the mutation causes large increases in the Michaelis constant indicating a reduced affinity for cytochrome c 685179, 685217 1.11.1.5 D34N surface mutant, shift in reduction potential to -175 mV. Analysis of spectroscopic properties 675241 1.11.1.5 D37E/P44D/V45D redesign of a manganese-binding site, ratio kcat/KM values for manganese oxidation is 0.33 per mM and s at pH 5.0 674935 1.11.1.5 D37E/V45E/H181E redesign of a manganese-binding site, ratio kcat/KM values for manganese oxidation is 0.25 per mM and s at pH 5.0 674935 1.11.1.5 D37K positive-to-negative charge-reversal mutant 685179 1.11.1.5 D37K the mutation causes large increases in the Michaelis constant indicating a reduced affinity for cytochrome c 685217 1.11.1.5 E117H no enzymatic activity 674744 1.11.1.5 E117K no enzymatic activity 674744 1.11.1.5 E117L no enzymatic activity 674744 1.11.1.5 E118K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E118K the mutation causes large increases in the Michaelis constant indicating a reduced affinity for cytochrome c 685217 1.11.1.5 E123D distal heme pocket mutant, no detectable electrocatalytic turnover of substrate in the highpotential regime 741922 1.11.1.5 E123Q distal heme pocket mutant, no detectable electrocatalytic turnover of substrate in the highpotential regime 741922 1.11.1.5 E17K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E201K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E209K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E290C formation of a covalent complex with cytochrome c mutant K79C, kinetic studies. Residual activity of complex is due to unreacted enzyme that copurifies with the complex. In the complex, the Pelletier-Kraut site is blocked which results in zero catalytic activity 672215 1.11.1.5 E290K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E290K the mutation causes large increases in the Michaelis constant indicating a reduced affinity for cytochrome c 685217 1.11.1.5 E290N surface mutant, shift in reduction potential to -177 mV. Analysis of spectroscopic properties 675241 1.11.1.5 E291K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E291Q surface mutant, shift in reduction potential to -162 mV. Analysis of spectroscopic properties 675241 1.11.1.5 E32K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E32Q surface mutant, shift in reduction potential to -168 mV. Analysis of spectroscopic properties 675241 1.11.1.5 E35K positive-to-negative charge-reversal mutant 685179 1.11.1.5 E98K positive-to-negative charge-reversal mutant 685179 1.11.1.5 F102W distal heme pocket mutant, 10fold decrease in peroxidase activity and high-potential catalytic turnover of hydrogen peroxide 741922 1.11.1.5 F81W modest changes in in vitro peroxidase assays 741922 1.11.1.5 G41E/V45E/H181D redesign of a manganese-binding site, ratio kcat/KM values for manganese oxidation is 0.10 per mM and s at pH 5.0 674935 1.11.1.5 G41E/V45E/W51F/H181D/W191F redesign of a manganese-binding site, ratio kcat/KM values for manganese oxidation is 0.6 per mM and s at pH 5.0 674935 1.11.1.5 G94K/K97Q/R100I site-directed mutagenesis, triple point mutant is created to mimic the critical loop region of, but its crystal structure reveals that the inactive, bishistidinyl-coordinated form of the active-site heme group is retained 705165 1.11.1.5 H52D distal pocket mutant, shift in reduction potential to -221 mV. Analysis of spectroscopic properties 675241 1.11.1.5 H52E distal pocket mutant, reduction potential -183 mV, comparable to wild-type 675241 1.11.1.5 H52K distal pocket mutant, shift in reduction potential to -157 mV. Analysis of spectroscopic properties 675241 1.11.1.5 H52L distal pocket mutant, shift in reduction potential to -170 mV. Analysis of spectroscopic properties 675241 1.11.1.5 H52L exhibits multiple forms in solution, with a reversible temperature-dependent interconversion, indicating the presence of a dynamic equilibrium between enzyme forms, which favors an apparent single form at low temperature and low pH, and a different form at high temperature and high pH 657944 1.11.1.5 H52L reacts with H2O2 at a lower rate 394630 1.11.1.5 H52L with slower cyanide dissociation rate constant for the heme group with respect to the wild-type enzyme 657943 1.11.1.5 H52L | site-directed mutagenesis, a distal pocket mutant -, 725645 1.11.1.5 H52L/W191F proximal pocket mutant, shift in reduction potential to -151 mV. Analysis of spectroscopic properties 675241 1.11.1.5 H52N | distal pocket mutant, shift in reduction potential to -259 mV, most negative reduction potential of all mutants analyzed. Analysis of spectroscopic properties 675241 1.11.1.5 H52Q distal pocket mutant, shift in reduction potential to -224 mV. Analysis of spectroscopic properties 675241 1.11.1.5 H52Q | site-directed mutagenesis, a distal pocket mutant -, 725645 1.11.1.5 H59G the distal His of the L-heme is removed 764157 1.11.1.5 H71G 55% activity compared to the wild type enzyme, contains a high-spin, presumably five-coordinate, peroxidatic heme site 679215 1.11.1.5 H71G about 55% of wild-type activity. Five-coordinate, peroxidatic heme structure contrary to six-coordinate structure of wild-type, formation of a tryptophan radical species during catalysis 679215 1.11.1.5 H71G the unactivated H71G mutant shows 75% of turnover activity of the wild-type enzyme in the activated form 688187 1.11.1.5 H71G/W94A 4% activity compared to the wild type enzyme, contains a high-spin, presumably five-coordinate, peroxidatic heme site 679215 1.11.1.5 H71G/W94A about 4% of wild-type activity. Five-coordinate, peroxidatic heme structure contrary to six-coordinate structure of wild-type, formation of a porphyrin radical species during catalysis 679215 1.11.1.5 H74M no enzymatic activity, reduced redox potential. The introduced methionine does not ligate the N-terminal heme 674744 1.11.1.5 H80G the distal His of the L-heme is removed 764157 1.11.1.5 H81G mutant is highly active 741922 1.11.1.5 H93G site-directed mutagenesis -, 724337 1.11.1.5 K12C characterization of complex with yeast cytochrome c mutant K79C. Cytochrome c is covalently bound and located 90° from its primary binding site. Catalytic activity is similar to wild-type cytochrome c peroxidase 672099 1.11.1.5 K149D positive-to-negative charge-reversal mutant 685179, 685217 1.11.1.5 K264C characterization of complex with yeast cytochrome c mutant K79C. Cytochrome c is covalently bound and located 90° from its primary binding site. Catalytic activity is similar to wild-type cytochrome c peroxidase 672099 1.11.1.5 M118H no enzymatic activity 674744 1.11.1.5 M118L 7.3% of wild-type activity 674744 1.11.1.5 M219Q/F247N site-directed mutagenesis 724366 1.11.1.5 M278H no enzymatic activity, reduced redox potential. Mutant contains two low-potential hemes 674744 1.11.1.5 M297H site-directed mutagenesis -, 724337 1.11.1.5 additional information a mutant lacking the putative cytochrome c peroxidase DocA shows a 10fold reduction in colonization of the chick cecum compared to wild-type enzyme, a mutant lacking the putative cytochrome c peroxidase CJJ0382 demonstrates a maximal 50fold colonization defect that is dependent on the inoculum dose -, 687074 1.11.1.5 additional information an unactivated mutant devoid of the protein loop shows 10% of turnover activity of the wild type enzyme in the activated form 688187 1.11.1.5 additional information construction of a DELTAccpA mutant Shewanella oneidensis line 724008 1.11.1.5 additional information construction of disruption knockout mutant DELTAZmcytC, phenotype, overview 725756 1.11.1.5 additional information construction of three apolar distal heme pocket mutants of CcP with altered pH dependencies compared to the wild-type enzyme 724463 1.11.1.5 additional information construction of three apolar distal heme pocket mutants of CcP with enhanced binding of 1-methoxynaphthalene near the heme and enhanced hydroxylation activity of 1-methoxynaphthalene 724658 1.11.1.5 additional information disruption and deletion mutants show intracellular growth defects in macrophage like cells in vitro. The enzyme provides protection against oxidative stress within macrophages in vitro -, 658807 1.11.1.5 additional information distal pocket mutants, proximal pocket mutants, channel mutants, surface mutations 394631 1.11.1.5 additional information generation of enzyme disruption mutant DELTAccp1, SOD2 activity is significantly lower in W191F ccp1 mutant cells than in DELTAccp1 deletion mutant cells -, 725070 1.11.1.5 additional information mutant with deletion of the translation start codon and 800 bp of the enzyme gene. Almost as active as the wild-type enzyme 657830 1.11.1.5 additional information pH dependence of the reduction potential and heme binding site structure analysis of wild-type and mutant enzymes using photoreduction and spectroscopic methods, respectively, overview -, 725645 1.11.1.5 additional information significant decreases in the rate of reaction with hydrogen peroxide with 56-, 300-, and 6200fold decreases for mutant (W51H), mutant (W51H/H52W), and mutant (W51H/H52L), respectively, compared to that of wild-type cytochrome c peroxidase, indicating that the position of the distal histidine has a significant effect on the rate of reaction with H2O2 702313 1.11.1.5 additional information variant of cytochrome c peroxidase in which the proposed electron transfer pathway is excised from the structure, leaving a water filled channel in its place 702250 1.11.1.5 N184R the N184R variant introduces potential hydrogen bonding interactions for ascorbate binding 702227 1.11.1.5 N184R/W191F site-directed mutagenesis 702227 1.11.1.5 N78C characterization of complex with yeast cytochrome c mutant K79C. Cytochrome c is covalently bound and located 90° from its primary binding site. Catalytic activity is similar to wild-type cytochrome c peroxidase 672099 1.11.1.5 P75T/H81K/E84Q site-directed mutagenesis 724366 1.11.1.5 Q107L no enzymatic activity 674744 1.11.1.5 Q113N distal heme pocket mutant, no detectable electrocatalytic turnover of substrate in the highpotential regime 741922 1.11.1.5 R31E positive-to-negative charge-reversal mutant 685179, 685217 1.11.1.5 R48A/W51A/H52A distal pocket mutant, shift in reduction potential to -163 mV. Analysis of spectroscopic properties 675241 1.11.1.5 R48A/W51A/H52A less than 0.02% of wild-type activity. The imidazole binding curve is biphasic. The fast phase of imidazole binding is linearly dependent on the imidazole concentration while the slow phase is independent of imidazole concentration. Imidazole binding is pH dependent with the strongest binding observed at high pH. Mutant displays higher binding affinities for 1-methylimidazole and 4-nitroimidazole than wild-type CcP 741980 1.11.1.5 R48A/W51A/H52A site-directed mutagenesis, the mutant has altered pKA values compred to the wild-type enzyme 724463 1.11.1.5 R48A/W51A/H52A site-directed mutagenesis, the mutant shows 34fold higher activity with 1-methoxynaphthalene than the wild-type enzyme. While wild-type CcP is very stable to oxidative degradation by excess hydrogen peroxide, mutant CcP is inactivated within four cycles of the peroxygenase reaction 724658 1.11.1.5 R48E distal pocket mutant, shift in reduction potential to -179 mV. Analysis of spectroscopic properties 675241 1.11.1.5 R48K distal pocket mutant, reduction potential -186 mV, comparable to wild-type. Analysis of spectroscopic properties 675241 1.11.1.5 R48K hexacoordinate, high-spin, unreactive against H2O2 394629, 394630 1.11.1.5 R48L distal pocket mutant, shift in reduction potential to -164 mV. Analysis of spectroscopic properties 675241 1.11.1.5 R48L reacts with H2O2 at a lower rate 394629, 394630 1.11.1.5 R48L/W51L/H52L distal pocket mutant, shift in reduction potential to -146 mV. Analysis of spectroscopic properties 675241 1.11.1.5 R48L/W51L/H52L less than 0.02% of wild-type activity. The imidazole binding curve is biphasic. The fast phase of imidazole binding is linearly dependent on the imidazole concentration while the slow phase is independent of imidazole concentration. Imidazole binding is pH dependent with the strongest binding observed at high pH. Mutant displays higher binding affinities for 1-methylimidazole and 4-nitroimidazole than wild-type CcP 741980 1.11.1.5 R48L/W51L/H52L site-directed mutagenesis, the mutant has altered pKA values compred to the wild-type enzyme 724463 1.11.1.5 R48L/W51L/H52L site-directed mutagenesis, the mutant shows higher activity with 1-methoxynaphthalene than the wild-type enzyme 724658 1.11.1.5 R48L/W51L/H52L | site-directed mutagenesis, a distal pocket mutant -, 725645 1.11.1.5 R48V/W51V/H52V distal pocket mutant, shift in reduction potential to -150 mV. Analysis of spectroscopic properties 675241 1.11.1.5 R48V/W51V/H52V less than 0.02% of wild-type activity. The imidazole binding curve is biphasic. Both phases have a hyperbolic dependence on the imidazole concentration. Imidazole binding is pH dependent with the strongest binding observed at high pH. Mutant displays higher binding affinities for 1-methylimidazole and 4-nitroimidazole than wild-type CcP 741980 1.11.1.5 R48V/W51V/H52V site-directed mutagenesis, the mutant has altered pKA values compred to the wild-type enzyme 724463 1.11.1.5 R48V/W51V/H52V site-directed mutagenesis, the mutant shows higher activity with 1-methoxynaphthalene than the wild-type enzyme 724658 1.11.1.5 S134P site-directed mutagenesis, distortion of the loop region, accompanied by an opening of the active-site loop, leaving the enzyme in a constitutively active state 705165 1.11.1.5 S134P/V135K site-directed mutagenesis, distortion of the loop region, accompanied by an opening of the active-site loop, leaving the enzyme in a constitutively active state 705165 1.11.1.5 V197C/C128A as active as the wild-type enzyme. Used to generate a covalent complex with a mutant cytochrome c 660372 1.11.1.5 V5C characterization of complex with yeast cytochrome c mutant K79C. Cytochrome c is covalently bound via disulfide formation of the mutated residues and located on the back-side of the enzyme, 180° from its primary binding site. Catalytic activity is similar to wild-type cytochrome c peroxidase. Significant electrostatic repulsion of the two cytochrome c molecules bound in an 2:1 complex which decreases as the ionic strength of buffer increases 672099 1.11.1.5 W191F catalytically inactive mature Ccp1 mutant, Ccp1W191F is a more persistent H2O2 signaling protein than wild-type Ccp1 -, 725070 1.11.1.5 W191F less efficient at catalytic turnover than the wild-type enzyme 659521 1.11.1.5 W191F mutation eliminates electron fast hole hopping through residue W191, enhancing accumulation of charge-separated intermediate and extending the timescale for binding/dissociation of the charge-separated complex. The photocycle includes dissociation/recombination of the charge-separated binary complex and a charge-separated ternary complex, [Zn-protoporphyrin+CcP, Fe2+cytochrome c, Fe3+cytochrome] 741907 1.11.1.5 W191F proximal pocket mutant, shift in reduction potential to -202 mV. Analysis of spectroscopic properties 675241 1.11.1.5 W191F reacts with H2O2 at a slightly higher rate 394630 1.11.1.5 W191F side chain replacement followed by four iterations of side chain sampling plus minimization of a region within 6 A of Trp191, in W191F partial formation of a covalent link from Trp51 to the heme is observed 702294 1.11.1.5 W191F site-directed mutagenesis 702227 1.11.1.5 W191F study of the role of intracomplex dynamics in controlling electron transfer, use of Zn-enzyme in 1:1 complex with cytochrome c 674164 1.11.1.5 W191G provides a specific site near heme from which substrates might be oxidized 394628 1.11.1.5 W191G the mutant exhibits a loop-gated artificial protein cavity 685084 1.11.1.5 W51F exhibits extensive dimerization 659521 1.11.1.5 W51H altered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rather than five-coordinate as it is in wild-type cytochrome c peroxidase, weaker effect on cyanide binding, with the cyanide affinity only 2-8times weaker than for cytochrome c peroxidase 702313 1.11.1.5 W51H distal pocket mutant, shift in reduction potential to -200 mV. Analysis of spectroscopic properties 675241 1.11.1.5 W51H/H52L altered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rather than five-coordinate as it is in wild-type cytochrome c peroxidase, weaker effect on cyanide binding, with the cyanide affinity only 2-8times weaker than for cytochrome c peroxidase 702313 1.11.1.5 W51H/H52L distal pocket mutant, shift in reduction potential to -162 mV. Analysis of spectroscopic properties 675241 1.11.1.5 W51H/H52W altered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rather than five-coordinate as it is in wild-type cytochrome c peroxidase, weaker effect on cyanide binding, with the cyanide affinity only 2-8times weaker than for cytochrome c peroxidase 702313 1.11.1.5 W94A less than 1% activity compared to the wild type enzyme, the mutant retains the normal six-coordinate heme structures 679215 1.11.1.5 W94A less than 1% of wild-type activity. Six-coordinate heme structure similar to wild-type 679215 1.11.1.5 W97A no enzymatic activity. W97 is the mediator of intramolecular electron transfer of the enzyme 674744 1.11.1.5 W97F no enzymatic activity. W97 is the mediator of intramolecular electron transfer of the enzyme 674744 1.11.1.5 Y229W mutant designed to incorporate a Trp-based extension to move oxidizing equivalents from the heme to the protein surface. Mutant is able to oxidize veratryl alcohol substrate with turnover numbers greater than wild type 742339 1.11.1.5 Y36A site-directed mutagenesis, Tyr36 directly blocks the equivalent ascorbate binding site in CcP and was therefore replaced with a less bulky residue 702227 1.11.1.5 Y36A/N184R site-directed mutagenesis, no significant spectroscopic changes on reaction with stoichiometric or higher amounts of H2O2 are seen 702227 1.11.1.5 Y36A/N184R/W191F site-directed mutagenesis, cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184, no evidence for a porphyrin pi-cation radical 702227 1.11.1.5 Y36A/W191F site-directed mutagenesis, no significant spectroscopic changes on reaction with stoichiometric or higher amounts of H2O2 are seen 702227 1.11.1.5 Y39A site-directed mutagenesis, mutation has a destabilizing effect on binding 705132