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Biomedical subjects

F Millett

Publications and source records attributed to F Millett.

At least 55 records · Page 3Linked to original sources
PubMed1987

The reaction of cytochromes c and c2 with the Rhodospirillum rubrum reaction center involves the heme crevice domain.

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 X 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 X 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.

Binding Sites↗
PubMed1987

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.

Adrenodoxin↗
PubMed1987

Reaction of cytochromes c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain.

In order to define the interaction domain on Rhodobacter sphaeroides cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged (carboxydinitrophenyl)- (CDNP-) lysines. The reaction mixture was separated into several different fractions by ion-exchange chromatography on (carboxymethyl)cellulose. Tryptic digests of these fractions were analyzed by reverse-phase peptide mapping to determine the lysines that had been modified. Fraction A was found to consist of a mixture of singly labeled derivatives modified at lysine-35, -88, -95, -97, and -105 and several other unidentified lysines comprising 32% of the total. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The second-order rate constant for the reaction of native cytochrome c2 with reaction centers was 2.0 X 10(8) M-1 s-1, while that for fraction A was 20-fold less, 1.0 X 10(7) M-1 s-1. This suggests that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice significantly lowered the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.

Amino Acid Sequence↗
PubMed1987

The binding domain on horse cytochrome c and Rhodobacter sphaeroides cytochrome c2 for the Rhodobacter sphaeroides cytochrome bc1 complex.

The interaction of the Rhodobacter sphaeroides cytochrome bc1 complex with Rb. sphaeroides cytochrome c2 and horse cytochrome c was studied by using specific lysine modification and ionic strength dependence methods. The rate of the reactions with both cytochrome c and cytochrome c2 decreased rapidly with increasing ionic strength above 0.2 M NaCl. The ionic strength dependence suggested that electrostatic interactions were equally important to the reactions of the two cytochromes, even though they have opposite net charges at pH 7.0. In order to define the interaction domain on horse cytochrome c, the reaction rates of derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were measured. Modification of lysine-8, -13, -27, -72, -79, and -87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This result indicates that lysines surrounding the heme crevice of horse cytochrome c are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. In order to define the reaction domain on cytochrome c2, a fraction consisting of a mixture of singly labeled 4-carboxy-2,6-dinitrophenylcytochrome c2 derivatives modified at lysine-35, -88, -95, -97, and -105 and several unidentified lysines was prepared. Although it was not possible to resolve these derivatives, all of the identified lysines are located on the front surface of cytochrome c2 near the heme crevice. The rate of reaction of this fraction was significantly smaller than that of native cytochrome c2, suggesting that the binding domain on cytochrome c2 is also located at the heme crevice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Fluorescein mercuric acetate specifically displaces zinc from cytochrome oxidase.

Treatment of beef heart cytochrome oxidase with fluorescein mercuric acetate (FMA) was found to specifically displace zinc from the enzyme and inhibit the steady-state activity in a parallel fashion. The native cytochrome oxidase preparation contained 2.3 Cu: 2.0 Fe: 1.1 Zn: 0.9 Mg. Addition of 2 equivalents of FMA inhibited the activity by 50% and displaced 60% of the zinc from the enzyme, but did not affect the copper, iron or magnesium content. The pre-steady-state reduction of cytochrome oxidase by ferrocytochrome c was not affected by the FMA treatment, in contrast to the inhibition of steady state activity. These results suggest a possible structural or functional role for zinc in cytochrome oxidase.

Animals↗
PubMed1986

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.

Adrenodoxin↗
PubMed1986

The use of a water-soluble carbodiimide to study the interaction between Chromatium vinosum flavocytochrome c-552 and cytochrome c.

The interaction between horse heart cytochrome c and Chromatium vinosum flavocytochrome c-552 was studied using the water-soluble reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Treatment of flavocytochrome c-552 with EDC was found to inhibit the sulfide: cytochrome c reductase activity of the enzyme. SDS gel electrophoresis studies revealed that EDC treatment led to modification of carboxyl groups in both the Mr 21 000 heme peptide and the Mr 46 000 flavin peptide, and also to the formation of a cross-linked heme peptide dimer with an Mr value of 42 000. Both the inhibition of sulfide: cytochrome c reductase activity and the formation of the heme peptide dimer were decreased when the EDC modification was carried out in the presence of cytochrome c. In addition, two new cross-linked species with Mr values of 34 000 and 59 000 were formed. These were identified as cross-linked cytochrome c-heme peptide and cytochrome c-flavin peptide species, respectively. Neither of these species were formed in the presence of a cytochrome c derivative in which all of the lysine amino groups had been dimethylated, demonstrating that EDC had cross-linked lysine amino groups on native cytochrome c to carboxyl groups on the heme and flavin peptides. A complex between cytochrome c and flavocytochrome c-552 was required for cross-linking to occur, since ionic strengths above 100 mM inhibited cross-linking.

Animals↗
PubMed1986

Complex formation and electron transfer between mitochondrial cytochrome c and flavocytochrome c552 from Chromatium vinosum.

Flavocytochrome c552 from Chromatium vinosum catalyzes the oxidation of sulfide to sulfur using a soluble c-type cytochrome as an electron acceptor. Mitochondrial cytochrome c forms a stable complex with flavocytochrome c552 and may function as an alternative electron acceptor in vitro. The recognition site for flavocytochrome c552 on equine cytochrome c has been deduced by differential chemical modification of cytochrome c in the presence and absence of flavocytochrome c552 and by kinetic analysis of the sulfide:cytochrome c oxidoreductase activity of m-trifluoromethylphenylcarbamoyl-lysine derivatives of cytochrome c. As with mitochondrial redox partners, interaction occurs around the exposed heme edge at the "front face" of cytochrome c. However, the domain recognized by flavocytochrome c552 seems to extend to the right of the heme edge, whereas the site of interaction with mitochondrial cytochrome c oxidase and reductase is more to the left. Km but not Vmax of the electron transfer reaction with mitochondrial cytochrome c increases with increasing ionic strength. The correlation of chemical modification and ionic strength dependence data indicates that the electrostatic interaction between the two hemoproteins involves fewer ionic bonds than that with other redox partners of cytochrome c.

Acetylation↗
PubMed1985

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate.

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

Animals↗
PubMed1985

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.

Amino Acid Sequence↗
PubMed1984

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.

Adrenodoxin↗
PubMed1984

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.

Adrenodoxin↗
PubMed1983

Enzymatic trimethylation of lysine-72 in cytochrome c.

The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowering the pI values of the protein than the chemical modification. Furthermore, the lowering of the pI value of cytochrome c by enzymatic methylation is highly dependent on the urea concentration. The presence of urea reduces the effect of methylation on the protein molecule and the difference in pI values virtually disappears with the increasing concentration of urea (6 M), which essentially disrupts the protein tertiary structure.

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

Cytochrome c is cross-linked to subunit II of cytochrome c oxidase by a water-soluble carbodiimide.

Modification of beef heart cytochrome c oxidase with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) or 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide (CH3EDC) has been found to significantly inhibit the high-affinity phase of the reaction of this enzyme with cytochrome c. Reaction conditions leading to a 70% inhibition of Vmax resulted in a 16-fold increase in the Km for cytochrome c. The loss in activity was accompanied by modification of subunit II to form a new species, II', which migrated somewhat more rapidly than the unmodified subunit during sodium dodecyl sulfate (NaDodSO4) gel electrophoresis. This new species was the major site of radiolabeling when cytochrome c oxidase was treated with [14C]CH3EDC, indicating covalent incorporation of the carbodiimide. Equimolar concentrations of cytochrome c dramatically protected cytochrome c oxidase from inhibition by the carbodiimide and in approximately the same proportion shielded subunit II from modification to the labeled II' species. In addition, cytochrome c was cross-linked to subunit II to form a new species migrating somewhat faster than subunit I during NaDodSO4 gel electrophoresis. This cross-linked species was shown to contain subunit II by using subunit-specific antibodies. We propose that EDC or CH3EDC reacts with one or more partially buried carboxyl groups on subunit II to form a positively charged N-acylurea which inhibits cytochrome c binding. In the presence of cytochrome c, EDC promotes formation of amide cross-links between lysine amino groups on cytochrome c and their complementary carboxyl groups on cytochrome c oxidase.

Animals↗