James E. Erman

Laue diffraction study on the structure of cytochrome c peroxidase compound I

BACKGROUND Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Yeast cytochrome c peroxidase: mechanistic studies via protein engineering

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.

Oxidation-reduction potential measurements of cytochrome c peroxidase and pH dependent spectral transitions in the ferrous enzyme

The redox potential of the ferrous/ferric couple in cytochrome c peroxidase has been measured as a function of pH between pH 4.5 and 8. The redox potential decreases linearly as a function of pH between pH 4.5 and 7 with a slope of --57 +/- 2 mV per pH unit. Above pH 7, there is a positive inflection in the midpoint potential versus pH plot attributed to an ionizable group in the ferrous enzyme with pKa of 7.6 +/- 0.1. The midpoint potential at pH 7 is--0.194 V relative to the standard hydrogen electrode at 25 degree C. Ferrocytochrome c peroxidase undergoes a reversible spectral transition as a function of pH. Below pH 7, the enzyme has a spectrum typical of high spin ferroheme proteins while above pH 8, the spectrum is typical of low spin ferroheme proteins. The transition is caused by a co-operative, two proton ionization with an apparent pKa of 7.7 +/- 0.2. Two other single proton ionizations cause minor perturbations to the spectrum of ferrocytochrome c peroxidase. One has a pKa of 5.7 +/- 0.2 while the second has a pKa of 9.4 +/- 0.2.

Axial histidyl imidazole non-exchangeable proton resonances as indicators of imidazole hydrogen bonding in ferric cyanide complexes of heme peroxidases.

Proton NMR spectra of a model of low-spin cyanide complexes of ferric hemoproteins indicate that two broad single-protein resonances from the axial imidazole can be resolved outside the diamagnetic spectral region. Upon deprotonation of the imidazole in the model, the upfield resonance shifts dramatically to higher field, suggesting that its position may reflect the degree of hydrogen bonding or proton donation of the imidazole. Met-cyano myoglobin reveals a pair of such broad peaks in the regions expected for an essentially neutral axial imidazole. In the cyano complexes of horseradish peroxidase and cytochrome c peroxidase, a pair of single-proton resonances are located which are assigned to the same imidazole protons on the basis of their linewidth and shift changes upon altering the heme substituents. The upfiled proton, however, is found at much higher field than in metMbCN. The upfield bias of this resonance is taken as evidence for appreciable imidazolate character for the axial ligand in these heme peroxidases.

A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes

Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and horse cytochrome c, but different from those reported for cytochrome c complexes with flavodoxin and cytochrome b5. By comparison with the shifts reported for lysine-13-modified cytochrome c we conclude that the results reported here support the Poulos-Kraut proposed structure for the molecular redox complex between cytochrome-c peroxidase and cytochrome c. These results indicate that the principal site of interaction with cytochrome-c peroxidase is the exposed heme edge of horse cytochrome c, in proximity to lysine-13 and the heme pyrrole II. The noncovalent cytochrome-c peroxidase-cytochrome c complex exists in the rapid-exchange time limit even at 500 mHz proton frequency. Our data provide an improved estimate of the minimum off-rate for exchanging cytochrome c as 1133 (+/- 120) s-1 at 23 degrees C.

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: A fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.

Heme accessibility in the ferricytochrome c-cytochrome c peroxidase complex

Complex formation between ferricytochrome c and cytochrome c peroxidase inhibits the rate of cyanide binding by ferricytochrome c nearly 90%. The reactions between cytochrome c peroxidase and fluoride or hydrogen peroxide are not significantly affected by complex formation with cytochrome c.

Assignment of hyperfine shifted resonances in high-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins

Assignments of hyperfine shifted proton resonances for the high-spin forms of cytochrome c peroxidase (EC 1.11.1.5) have been made (cytochrome c peroxidase, cytochrome c peroxidase-F) employing the technique of reconstituting the apoprotein with specifically deuterated protohemin IX derivatives. The results show that the heme methyl group pattern differs significantly from similar assignments made for metmyoglobin. In cytochrome c peroxidase the methyl pattern is 5 greater than 1 greater than 8 greater than 3. For cytochrome c peroxidase-F the pattern is 5 greater than 8 greater than 1 greater than 3, but the resonances are not shifted as far downfield and they exhibit a narrower spread. For myoglobin the relative methyl ordering has previously been shown to be 8 greater than 5 greater than 3 greater than 1. Several conclusions have been reached, including confirmation of the essential correspondence between the solution- and crystal-derived data for several heme crevice structural features. The pH dependence of the cytochrome c peroxidase-F methyl resonances is also presented and is shown to differ from native peroxidase. For cytochrome c peroxidase-F smooth, continuous titrations are observed with no evidence of the second conformation which was found for the native enzyme.

Proton magnetic resonance studies of cytochrome c peroxidase: pD dependence of the isotropically shifted resonances

Abstract Proton nuclear magnetic resonance studies of the isotropically shifted resonances of native cytochrome c peroxidase have been carried out at 360 MHz. Proton resonances extend to 84 ppm downfield and 12 ppm upfleld from 2,2-dimethyl-2-silapentane-5-sulfonate and are characteristic of high-spin iron +3 heme proteins. Between pH 8 and 4.1 the isotropic resonances exhibit dramatic pH-dependent behavior which demonstrates the presence of two acid-base interconversions. One process, with a p K a of 5.8, is slow on the NMR time scale and probably represents a protein conformation change. This process correlates with an apparent p K a observed in the kinetic properties of the enzymes, with the alkaline form being the enzymatically active species. A second ionization with a p K of 4.9 is fast on the NMR time scale and probably represents a true ionization.

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties.

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20-40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 +/- 5 s-1; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s-1 can be estimated at 5 degrees C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10(-4) to 10(-5)-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:1 covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.