Incorporation of galactose into galactosyltransferase.

Bovine skim milk galactosyltransferase (EC 2.4.1.22) retained its catalytic activity after partial enzymatic removal of sialic acid and galactose. Desialylated and degalactosylated galactosyltransferase was a galactosyl acceptor in the galactosyltransferase reaction. [14C]Galactose from UDP-[14C]galactose was incorporated into the carbohydrate-depleted galactosyltransferase by native galactosyltransferase. The results suggest that galactosyltransferase participates in the biosynthesis of its glycopeptides of the sialic acid-galactose-N-acetylglucosamine type.

The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin.

A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, has been used to cross-link horse heart cytochrome c to spinach chloroplast plastocyanin. The complex was formed in yields up to 90% and was found to have a stoichiometry of 1 mol plastocyanin per mol cytochrome c. The cytochrome c in the complex was fully reducible by ascorbate and potassium ferrocyanide, and had a redox potential only 25 mV less than that of native cytochrome c. The complex was nearly completely inactive towards succinate-cytochrome c reductase and cytochrome c oxidase, suggesting that the heme crevice region of cytochrome c was blocked. We propose that the carbodiimide promoted the formation of amide cross-links between lysine amino groups surrounding the heme crevice of cytochrome c and complementary carboxyl groups on plastocyanin. It is of interest that the high-affinity site for cytochrome c binding on bovine heart cytochrome c oxidase has recently been found to involve a sequence of subunit II with some homology to the copper-binding sequence of plastocyanin.

Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase.

A new technique has been introduced to measure interprotein electron transfer which involves photoexcitation of a tris(bipyridine)ruthenium (Ru) complex covalently attached to one of the proteins. Four different strategies have been developed to specifically attach Ru to protein lysine amino groups, histidine imidazole groups, and cysteine sulhydryl groups. These strategies have been used to prepare more than 20 different singly-labeled Ru-cytochrome c derivatives. The new ruthenium photoexcitation technique has been used to study the mechanism for electron transfer between cytochrome c and cytochrome c peroxidase. Laser excitation of a complex between Ru-cytochrome c and cytochrome c peroxidase compound I results in formation of Ru(II*) which is a strong reducing agent, and rapidly transfers an electron to heme c Fe(III) to form Fe(II). The heme c Fe(II) then rapidly transfers an electron to the Trp-191 radical cation in CMPI. The rate constant for this reaction is 6 x 10(4) s-1 for a horse Ru-cytochrome c derivative labeled at lysine 27, and greater than 10(6) s-1 for yeast Ru-cytochrome c derivatives. A second laser flash results in electron transfer from heme c to the oxyferryl heme in cytochrome c peroxidase compound II with a rate constant of 350 s-1. The ruthenium photoreduction technique has been used to study the interaction domain between the two proteins, the pathway for electron transfer to the radical cation and the oxyferryl heme, and the specific residues in the heme crevice which control the electron transfer properties of the Trp-191 radical cation and the oxyferryl heme.

Double-pionic fusion of nuclear systems and the "ABC" effect: approaching a puzzle by exclusive and kinematically complete measurements.

The ABC effect-a puzzling low-mass enhancement in the pipi invariant mass spectrum, first observed by Abashian, Booth, and Crowe-is well known from inclusive measurements of two-pion production in nuclear fusion reactions. Here we report on the first exclusive and kinematically complete measurements of the most basic double-pionic fusion reaction pn-->dpi;{0}pi;{0} at beam energies of 1.03 and 1.35 GeV. The measurements, which have been carried out at CELSIUS-WASA, reveal the ABC effect to be a (pipi)_{I=L=0} channel phenomenon associated with both a resonancelike energy dependence in the integral cross section and the formation of a DeltaDelta system in the intermediate state. A corresponding simple s-channel resonance ansatz provides a surprisingly good description of the data.

Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding.

Treatment of cytochrome P-450scc with fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 +/- 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 +/- 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.

Interaction between adrenodoxin and cytochrome c.

The reduction of horse heart ferricytochrome c by reduced adrenodoxin was found by stopped flow spectroscopy to follow second order kinetics with a rate constant of 7.8 X 10(6) M-1 s-1 in Tris/Cl, pH 7.5, at an ionic strength of 0.2 M, 29 degrees C. The temperature dependence of the rate constant was used to determine that the activation parameters were delta H++++ = 7.7 kcal/mol and delta S = -1.5 cal/mol degrees K. The rate constant decreased rapidly with increasing ionic strength, indicating that electrostatic interactions between the two proteins were important to the reaction. The contribution of individual lysine amino groups to the electrostatic interaction was determined by measuring the reaction rate of specifically trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives. Modification of lysines 13, 27, 72, and 79 surrounding the heme crevice decreased the reaction rate by about 2-fold, while modification of lysine amino groups in other regions of cytochrome c had decreasing effects as the distance from the heme crevice was increased. The interaction domain therefore involves specific complementary charge interactions between lysine amino groups immediately surrounding the heme crevice of cytochrome c and carboxylate groups on adrenodoxin. A semi-empirical relationship was developed for the total electrostatic interaction between the two proteins which is in quantitative agreement with both the ionic strength dependence of the reaction rate and the specific lysine modification studies.

Adrenodoxin interaction with adrenodoxin reductase and cytochrome P-450scc. Cross-linking of protein complexes and effects of adrenodoxin modification by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Modification of the three carboxyl groups on adrenodoxin using a water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) caused a weakening of the binding of this iron-sulfur protein to both its electron donor protein, adrenodoxin reductase, and its electron acceptor protein, cytochrome P-450scc. Based upon the proximity of the modified groups, the site on adrenodoxin for interaction with the other two proteins is likely to be either identical or highly overlapping, and formation of a ternary complex among the proteins is precluded. Upon incubation of adrenodoxin and either adrenodoxin reductase or cytochrome P-450 plus the carbodiimide (1:1), covalently cross-linked species were formed. When all three proteins were incubated with the cross-linker, only the binary complexes were formed, and no higher order (e.g. 1:1:1 or 1:2:1) complexes were seen. These studies indicate that adrenodoxin forms exclusive binary complexes with its electron transfer partner proteins, and thus provide a physical explanation for the proposed role of adrenodoxin as a mobile electron shuttle between NADPH-adrenodoxin reductase and cytochrome P-450scc.

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc.

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.

Photoinduced electron transfer between cytochrome c peroxidase and yeast cytochrome c labeled at Cys 102 with (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]ruthenium2+.

The synthesis of (4-bromomethyl-4'-methylbipyridine) [bis(bipyridine)]ruthenium(II) hexafluorophosphate is described. This new reagent was found to selectively label the single sulfhydryl group at Cys-102 on yeast iso-1-cytochrome c to form the (dimethylbipyridine-Cys-102-cytochrome c)[bis(bipyridine)]ruthenium derivative (Ru-102-cyt c). Excitation of Ru-102-cyt c with a short light flash resulted in formation of excited-state Ru(II*), which rapidly transferred an electron to the ferric heme group to form Fe(II). When the cytochrome c peroxidase compound I (CMPI) was present in the solution, electron transfer from photoreduced Fe(II) in Ru-102-cyt c to the radical site in CMPI was observed. At high ionic strength (100 mM sodium phosphate and 25 mM EDTA, pH 7), second-order kinetics were observed with a rate constant of (7.5 +/- 0.7) x 10(7) M-1 s-1. The second-order rate constant for native iso-1-cytochrome c was (6.7 +/- 0.7) x 10(7) M-1 s-1 under these conditions. The second-order rate constant for electron transfer from Ru-102-cyt c to the radical site in CMPI increased as the ionic strength was decreased, reaching a value of (4.8 +/- 0.5) x 10(8) M-1 s-1 in 40 mM EDTA, pH 7. At lower ionic strength, a complex was formed between Ru-102-cyt c and CMPI, and the rate for intracomplex electron transfer to the radical site was found to be greater than 50,000 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron transfer between cytochrome c and cytochrome c peroxidase.

The reaction between cytochrome c (CC) and cytochrome c peroxidase (CcP) is a very attractive system for investigating the fundamental mechanism of biological electron transfer. The resting ferric state of CcP is oxidized by hydrogen peroxide to compound I (CMPI) containing an oxyferryl heme and an indolyl radical cation on Trp-191. CMPI is sequentially reduced to CMPII and then to the resting state CcP by two molecules of CC. In this review we discuss the use of a new ruthenium photoreduction technique and other rapid kinetic techniques to address the following important questions: (1) What is the initial electron acceptor in CMPI? (2) What are the true rates of electron transfer from CC to the radical cation and to the oxyferryl heme? (3) What are the binding domains and pathways for electron transfer from CC to the radical cation and the oxyferryl heme? (4) What is the mechanism for the complete reaction under physiological conditions?

Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases.

Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.

Identification of specific carboxylate groups on adrenodoxin that are involved in the interaction with adrenodoxin reductase.

Modification of bovine adrenodoxin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dramatically inhibited the reaction with adrenodoxin reductase (EC 1.18.1.2). The modification did not cause any change in the visible spectrum of adrenodoxin, indicating that the iron-sulfur center was not perturbed. Furthermore, the anomalous fluorescence of Tyr 82 was not changed in either intensity or wavelength. The inhibition was accompanied by the covalent incorporation of 14C-labeled EDC into adrenodoxin. The sites modified by EDC were determined by hydrolyzing adrenodoxin with either trypsin or Staphylococcus aureus protease and separating the resulting peptides by reverse phase high pressure liquid chromatography. The major carboxyl groups modified were found to be at Glu 74, Asp 79, and Asp 86, which are located in a sequence containing a high negative charge density. We propose that the conversion of negatively charged carboxylate groups at these residues to bulky, positively charged EDC-carboxyl groups inhibits the reaction with the reductase. EDC was also found to cross-link adrenodoxin to cytochrome c in yields up to 90%. The cross-links were found to involve the formation of amide linkages between carboxyl groups on adrenodoxin and the lysine amino groups surrounding the heme crevice of cytochrome c.

Reaction of horse cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I.

The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M-1 s-1. The rate constant for the reduction of the radical is nearly independent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase.

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.

Identification of the binding site on cytochrome c1 for cytochrome c.

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.

Interaction domain for the reaction of cytochrome c with the radical and the oxyferryl heme in cytochrome c peroxidase compound I.

Site-directed mutants of cytochrome c peroxidase (CcP) were created to modify the interaction domain between CcP and yeast iso-1-cytochrome c (yCC) seen in the crystal structure of the CcP-yCC complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. In the crystalline CcP-yCC complex, two acidic regions of CcP contact lysine residues on yCC. Mutants E32Q, D34N, E35Q, E290N, and E291Q were used to examine the effect of converting individual carboxylate side chains in the acidic regions to amides. The A193F mutant was used to test the effect of introducing a phenyl moiety at the point of closest contact between CcP and yCC in the crystal structure. Stopped-flow experiments carried out in 310 mM ionic strength buffer at pH 7 revealed that yCC initially reduced the indole radical on Trp-191 of the parent CcP compound I with a bimolecular rate constant ka = 2.5 x 10(8) M-1 s-1. A second molecule of yCC subsequently reduced the oxyferryl heme of compound II with a rate constant kb = 5 x 10(7) M-1 s-1. The bimolecular rate constants ka and kb were affected in parallel by each mutation examined. CcP mutants D34N and E290N that are closest to a complementary yCC lysine residue in the crystalline CcP-yCC complex gave the lowest values for ka and kb, which were 25-50% of the values of the CcP parent. Mutants E32Q and E291Q that are removed from the interaction domain gave the same ka and kb values as the CcP parent.(ABSTRACT TRUNCATED AT 250 WORDS)

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. Ii. Rapid kinetic analysis of electron transfer from cytochrome c to Rhodobacter sphaeroides cytochrome oxidase surface mutants.

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Role of methionine 230 in intramolecular electron transfer between the oxyferryl heme and tryptophan 191 in cytochrome c peroxidase compound II.

The kinetics of electron transfer from cytochrome c (CC) to yeast cytochrome c peroxidase (CcP) compound I were studied by flash photolysis and stopped-flow spectroscopy. Flash photolysis studies employed horse CC derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis-(bipyridine)ruthenium (Ru-CC). Initial electron transfer from Ru-CC reduced the indole radical on Trp-191 of CcP compound I [CMPI(IV,R.)], producing CMPII(IV,R). This reaction was biphasic for each of several Ru-CC derivatives, with rate constants which varied according to the position of the Ru label. For Ru-27-CC labeled at lysine 27, rate constants of 43,000 and 1600 s-1 were observed at pH 5.0 in 2 mM acetate. After reduction of the indole radical by Ru-CC, intramolecular electron transfer from Trp-191 to the oxyferryl heme in CMPII(IV,R) was observed, producing CMPII(III,R.). The rate constant and extent of this intramolecular electron transfer reaction were independent of both the protein concentration and the Ru-CC derivative employed. The rate constant decreased from 1100 s-1 at pH 5 to 550 s-1 at pH 6, while the extent of conversion of CMPII(IV,R) to CMPII(III,R.) decreased from 56% at pH 5 to 29% at pH 6. The reaction was not detected at pH 7.0 and above. The pH dependence of the rate and extent of this internal electron transfer reaction paralleled the pH dependence of the rate of bimolecular reduction of CMPII(IV,R) by native horse CC measured by stopped-flow spectroscopy at high ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase.

A new ruthenium-labeled cytochrome c derivative was designed to measure the actual rate of electron transfer to the Trp-191 radical cation and the oxyferryl heme in cytochrome c peroxidase compound I {CMPI(FeIV = O,R.+)}. The H39C,C102T variant of yeast iso-1-cytochrome c was labeled at the single cysteine residue with a tris (bipyridyl)ruthenium(II) reagent to form Ru-39-Cc. This derivative has the same reactivity with CMPI as native yCc measured by stopped-flow spectroscopy, indicating that the ruthenium group does not interfere with the interaction between the two proteins. Laser excitation of the 1:1 Ru-39-Cc-CMPI complex in low ionic strength buffer (2 mM phosphate, pH 7) resulted in electron transfer from RuII* to heme c FeIII with a rate constant of 5 x 10(5) s-1, followed by electron transfer from heme c Fe II to the Trp-191 indolyl radical cation in CMPI(FeIV = O,R*+) with a rate constant of k(eta) = 2 x 10(6) s-1. A subsequent laser flash led to electron transfer from heme c to the oxyferryl heme in CMPII-(FeIV = O,R) with a rate constant of k(etb) = 5000 s-1. The location of the binding domain was determined using a series of surface charge mutants of CcP. The mutations D34N, E290N, and A193F each decreased the values of k(eta) and k(etb) by 2-4-fold, consistent with the use of the binding domain identified in the crystal structure of the yCc-CcP complex for reduction of both redox centers [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. A mechanism is proposed for reduction of the oxyferryl heme in which internal electron transfer in CMPII(FeIV = O,R) leads to the regeneration of the radical cation in CMPII-(FeIII,R*+), which is then reduced by yCcII. Thus, both steps in the complete reduction of CMPI involve electron transfer from yCcII to the Trp-191 radical cation using the same binding site and pathway. Comparison of the rate constant k(eta) with theoretical predictions indicate that the electron transfer pathway identified in the crystalline yCc-CcP complex is very efficient. Stopped-flow studies indicate that native yCcII initially reduces the Trp-191 radical cation in CMPI with a second-order rate constant ka, which increases from 1.8 x 10(8) M-1 s-1 at 310 mM ionic strength to > 3 x 10(9) M-1 s-1 at ionic strengths below 100 mM. A second molecule of yCcII then reduces the oxyferryl heme in CMPII with a second-order rate constant kb which increases from 2.7 x 10(7) M-1 s-1 at 310 mM ionic strength to 2.5 x 10(8) M-1 s-1 at 160 mM ionic strength. As the ionic strength is decreased below 100 mM the rate constant for reduction of the oxyferryl heme becomes progressively slower as the reaction is limited by release of the product yCcIII from the yCcIII-CMPII complex. Both ruthenium photoreduction studies and stopped-flow studies demonstrate that the Trp-191 radical cation is the initial site of reduction in CMPI under all conditions of ionic strength.