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Research has been conducted on the Saccharomyces cerevisiae cytochrome c peroxidase. The overexpression of the forms of this peroxidase in Escherichia coli by growth in the deuterated medium has been carried out and reported.

Research was conducted to investigate the role of distal histidine on the reaction of cytochrome c peroxidse (Ccp) and its mutant form with neutral and anionic forms of p-nitroperoxybenzoic (pNPBA) acid. Results show that both forms of pNPBA react with Ccp at a faster rate than with the mutant form suggesting that histidine participates in more than a simple base catalysis reaction.

Results indicate that the reaction rates are influenced by protein heme group ionizations and are independent of ionic strength suggesting that the rate-limiting step is governed by reactants diffusion through the protein matrix.

Proton hyperfine resonance assignments for wild-type yeast iso-1-ferricyt c in the free state and complexed state were made. Proteins in their paramagnetic state were used. Proton resonance assignments for hyperfine-shifted protons of amino acids near the heme allowed identification of complex-induced proton shifts in CcP/cytochrome c complexes. The iso-1-ferricytochrome c magnetic axis system orientation shifts was found significant during complex formation.

The effects of the negatively charged sites within the cytochrome c on the surface of cytochrome c peroxidase were analyzed to determine the interaction between horse cytochrome c and CcP. The strong ionic strength dependence of the interaction between CcP and cytochrome c indicates that the binding is largely electrostatic in nature. Furthermore, the structure of the CcP/horse cytochrome c complex in solution was similar to its structure in the crystalline state.

The imidazole group of His at position 52 in the yeast enzyme, cytochrome c peroxidase, CcPCN, is necessary for catalysis. NMR spectroscopic studies of a mutant in which Asn82 is replaced with Ala indicate that the mutation disrupts a hydrogen bond between His52 and the heme-ligated CN. The heme active site of the enzyme appears to be microheterogenous. The enzyme preparation has two magnetically inequal forms that interconvert with changes in temperature. The equilibrium constant of the enzyme is 1.5 at 20 degrees celsius.

The Trp 191 to Phe mutant enzyme cytochrome c peroxidase-(MI,F191) (CcP) catalyzes the steady-state oxidation of Cc2+ avoiding reduction of the oxy-ferryl heme. The rise in steady-state functioning of the Trp 191 to Phe enzyme through second-site mutations is possible by enhancing either the reactivity of the oxy-ferryl enzyme with peroxide or the reactivity of Cc2+ with the oxidized intermediates formed during the reaction of oxy-ferryl enzyme with peroxide.

The steady-state kinetic studies of cytochrome c peroxidase (CcP) show that the cytochrome c peroxidase compound I (CcP-I) does not participate in Trp-191 radical formed during the reaction between the CcP and hydrogen peroxide. This CcP-I(sub A) form is directly reduced to cytochrome c peroxidase compound II CcP-II(sub F) by ferrocytochrome c. The steady-state velocity is seen when the rate of reduction of CcP-I(sub A) and CcP-II(sub F) is same as the rate of reduction of CcP-I.

The oxidation of yeast takes place at the high-affinity binding sites on cytochrome c peroxidase (CcP). The high-affinity binding sites increase their ionic strength from 0.010 to 0.20 M. The cytochrome c peroxidase catalyzes the oxidation of yeast iso-1 ferrocytochrome c by utilizing hydrogen peroxide. The two oxidized intermediates of CcP compound I and II to couple the reduction of two radicals of hydrogen peroxidase are in the ratio 2:1.

The observed enthalpy change remains static over the same range of ionic strength when horse ferricytochrome c binds to yeast cytochrome c peroxidase. Complex formation at 0.0100 M ions strength is followed by the discharge of 0.54 plus or minus 0.11 proton which varies according to the buffer. The corrected values for the enthalpy are 2.84 plus or minus 0.26 kilo calorie/mol. The entropy correction is plus 21 plus or minus 3 eu as is revealed by protein binding to the buffer.

A study of protein-protein association by hydrogen-deuterium exchange labeling and proton NMR using cytochrome c peroxidase (CCP) and yeast iso-1 ferricytochrome c reveals that exchange rates of backbone amide protons of yeast iso-1 ferricytochrome c become slow due to complex formation with CCP. The back region of iso-1 ferricytochrome c is involved in CCP binding. CCP binding may result in conformational change in iso-1 ferricytochrome c.

HNMR spectroscopic studies of the dynamics of yeast isozyme-1 ferricytochrome c equilibrium exchange in non-covalent complexes containing a 1:1 ratio of two yeast cytochrome c peroxidases (CcP) reveals that the exchange occurs between the P-bound and free environments. The exchange ranges between slow and intermediate on the NMR time scale. The preexchange lifetimes of free and bound iso-1 ferricytochrome c depend on saturation and inversion transfers.

Cyanide-ligated cytochrome C peroxidase (CcP(N), horse ferricyte C and yeast isozyme-1 ferricyt C) reveals proton NMR spectra that show prominent modifications due to the stimulation by production a complex. Complexes with resting-state Ccp and ferricytochromes C show similar alterations. The heme active site of CcPCN is the destination of the transmission of ferricyte C binding. The solution-state conformation of the complexes resembles the pattern of shifts stimulated by heme pyrrole C replacements and the heme pyrrole. A replacements yield important CcPCN NMR shifts that are stimulated by complexes.

Two cytochrome c peroxidase (CcP) mutants, CcP(R48L) and CcP(R48K), are examined on the basis of their crystallographic conformations. A range between pH 4 and 8 is adopted to study the electronic absorption spectrum and the hydrogen peroxide reactivity of the two mutants. The range between pH 4 and 6.5 displays a separate pentacoordination of the heme iron which resembles the crystallographic geometry and the electronic absorption spectrum of CcP(R48L). An increase in the pH level leads to the creation of an alkaline bis-imidazole form of CcP from CcP(R48L), which corresponds between the distal histidine and the heme iron.

The substitution of His-52 with Len is observed in a recombinant Cytochrome c peroxidase (CcP(MI)) expressed in Escherichia coli mutant, and its speed and conformation are determined. Speed of the reaction with peroxide is attenuated by a factor of 10 to the power 5 due to the replacement, and is associated with the CcP(MI) parent. The speed of the replacement of Arg-48 with Leu is higher than that of the reaction with peroxide, which resembles that of the interaction between metymyoglobin and hydrogen peroxide. CcP(MI) activates hydrogen peroxide principally due to the distal histidine residue, His-52, which makes the oxidized intermediate constant.

A study was conducted on the influence of the substitution of an asparagine residue for Asp-235 on mutant cytochrome c peroxidase. The substitution was found to have effects on the absoprtion spectrum and hydrogen peroxide reactivity of the peroxidase. Results revealed that the mutation/substitution radically changes the ligation at the sixth coordination site of the heme iron.

Four different noncovalent complexes and two covalently cross-linked complexes of cytochrome c peroxidase (CcP) were examined and compared using proton nuclear magnetic resonance spectroscopy. The noncovalent complexes were isolated from horse, tuna, yeast isozyme-1 and yeast isozyme-2 CcPs while the covalent cross-linked complexes were isolated from CcPs of horse and yeast isozyme-1 ferricytochromes c. The noncovalent complexes formed between CcP and their redox partners were noted to be dissimilar with those formed between CcP and ferricytochromes c from horse and tuna.

Evidence for complex formation in noncovalent cytochrome c peroxidase (CcP)-CN/ferricytochrome c complexes is presented. NMR studies show that both CcP-CN and iso-1-ferricytochrome c heme methyl resonances have large shifts arising from complex formation. The heme 3-methyl resonance shifts 0.166 ppm to lower frequency, while the CcP-CN heme 8-methyl resonance shifts 0.547 ppm to lower frequency.

Three cytochrome c peroxidase (CcP) mutants in which tryptophan 51 was replaced with a histidine residue were constructed to explore the effect of distal histidine position on the properties of (CcP). All three mutants, CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L) were observed to have altered electronic absorption spectra and form a CcP compound I-like intermediate, in which the Fe(IV) site decays between 500 and 3000 times more rapidly than the Fe(IV) site in wild-type CcP Compound I.

The impact of different values of pH and alkaline denaturation on the cyanide binding of the direct oxygen sensor from Escherichia coli (EcDosH) to the ferric heme domain is studied. The apparent association rate constant for cyanide binding to EcDosH is shown to increase linearly with the pH.

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) were constructed and characterized to determine the effect of localized charge on the catalytic properties of the enzyme CcP. The crystal structures of the cytochrome c/CcP complex provide evidence for a single CcP, catalytically active, cytochrome c binding domain.

The characterization and the mass instabilities of the various isolated recombinant Bradyrhizobium japonicum FixL heme domains (BjFixLH) are discussed. The intact heme domains gave normal UV-visible spectra, thus indicating that they are correctly assembled.

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) are constructed to examine the effect of localized charge on the catalytic properties of the enzyme. The results have shown that five of the 15 mutations cause large increases in the Michaelis constant, thus indicating that the cytochrome c-CcP complex is the dominant catalytically active complex in solution.

Specifically designed protein mutants were used to synthesize four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) via disulfide bond formation. The data provide evidence for significant electrostatic repulsion between bound cytochrome c molecules on the surface of CcP at low ionic strength with the interaction energy diminishing with increasing ionic strength.

A refinement of the synthesis and purification of the covalent complexes described by Papa and Poulos is described and a demonstration that the residual activity reported for the CcP(E290C)-cytochrome c(K73C) complex is due to small amounts of free CcP that copurify with the covalent complex. A demonstration that the covalent complex, in which the Pelletier-Kraut crystallographic site is blocked, is completely inactive is presented by measuring the activity of fractions containing the covalent complex during a gradient separation procedure.

The absorption spectrum of cytochrome c peroxidase in which His-52 is replaced with lysine residue CcP(H52K) and the reaction with hydrogen peroxide and HCN between pH 4 and 8 are studied. The results show that the unprotonated form, Lys-52 acts as a base catalyst facilitating the reactions of both hydrogen peroxide and HCN with CcP(H52K), as CcP(H52K) reacts with hydrogen peroxide at a slower rate than that of wild-type CcP but reacts with HCN at a faster rate than the wild-type enzyme.

Results show that the mutant cytochrome c peroxidase (H52L) preferentially binds the cyanide anion in contrast to the wild-type enzyme which binds hydrogen cyanide. Data indicate that above pH 8, the mutant enzymes exhibits faster cyanide binding. Furthermore, cyanide dissociates 4 times slower from the mutant enzyme.

Results reveal the mutant form of cytochrome c peroxidase (H52L) exhibit multiple enzyme forms in solution as opposed to the wild-type, which exists in a single form. The resting-state recombinant mutant enzyme shows two forms, which inteconvert depending on the solution temperature. Futhermore, the cyanide-ligated recombinant mutant enzyme shows 4 distinct enzyme forms, which interconvert with changing pH, temperature, and solven isotope composition.

The catalysis of cytochrome c peroxidase (CcP) reduction of hydrogen peroxide to water, with the use of two reducing equivalents from ferrocytochrome, involves the 2-equioxidation of the native Fe(III) enzyme to a higher oxidation state called Ccp compound I.

The crystallographic structures of the active-site cytochrome c peroxidase mutants, CcP(W51F), CcP(W191F) and CcP(D235N), reveal that these mutants exhibit small and localized structural perturbations. The replacement of various proximal and distal sites apparently cause the occurrence of these disruptions. Moreover, the study indicates that replacement of His-52 site lowers the hydrogen peroxide reaction rate by five orders of magnitude. In addition, Arg-48 is found to promote stabilization of cytochrome c peroxidase compound I.