Effect of the hinge protein on the heme iron site of cytochrome c1

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].     

A mitochondrial protein essential for the formation of the cytochrome c1-c complex. Isolation, purification, and properties

A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.     

Electron transfer between liposomal cytochrome c1 and cytochrome c: catalytic implications of electrostatic potentials

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.     

Effect of pyridoxal phosphate on the formation of cytochrome c1-c and cytochrome c-cytochrome oxidase complexes.

The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Role of the hinge protein in the electron transfer between cardiac cytochrome c1 and c. Equilibrium constants and kinetic probes

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.     

A cytochrome c methyltransferase from Crithidia oncopelti.

The mitochondrial cytochrome c-557 of Crithidia oncopelti contains two lysine residues and an N-terminal proline residue that are methylated in vivo by the methyl group of methionine. The purified cytochrome can act as a methyl acceptor for a methyltransferase activity in the cell extract that uses S-adenosylmethionine as methyl donor. Crithidia cytochrome c-557 is by far the best substrate for this methyltransferase of those tested, in spite of the fact that methylation sites are already almost fully occupied. The radioactive uptake of [14C]methyl groups from S-adenosylmethionine occurred only at a lysine residue (-8) and the N-terminal proline residue. This methyltransferase appears to differ from that of Neurospora and yeast [Durban, Nochumson, Kim, Paik & Chan (1978) J. Biol. Chem. 253, 1427-1435; DiMaria, Polastro, DeLange, Kim & Paik (1979) J. Biol. Chem. 254, 4645-4652] in that lysine-72 of horse cytochrome c is a poor acceptor. Also, the Crithidia methyltransferase appears to be stable to carry lysine methylation much further to completion than do the enzymes from yeast and Neurospora, which produce very low degrees of methylation in native cytochromes c.     

The oxygen reactions of reduced cytochrome c oxidase. Position of a form with an unusual EPR signal in the sequence of early intermediates.

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637--6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 196 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as 'Compound A' and 'Compound B' (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226--9237). Its appearance is within the time range reported for the formation of 'oxygenated' cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King, T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331--340, Elsevier/North-Holland Biomedical Press, Amsterdam).     

Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide's sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J.L., Slaughter, C.A., & Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., & Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambiguous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.     

Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol

Mucidin and strobilurin A, antifungal antibiotics isolated from the basidiomycetes Oudemansiella mucida and Strobiluris tenacellus, respectively, inhibit electron-transfer reactions in the cytochrome bc1 complex of the mitochondrial respiratory chain. The two compounds have identical effects on oxidation-reduction reactions of the cytochromes b and c1 in isolated succinate-cytochrome c reductase. They inhibit reduction of cytochrome c1 by succinate but do not inhibit reduction of cytochrome b. When added in combination with antimycin, either inhibitor blocks reduction of both cytochromes b and c1. Mucidin and strobilurin A differ from antimycin in that they inhibit, rather than promote, oxidant-induced reduction of cytochrome b. They also differ from antimycin in that they do not block reduction of cytochrome b by succinate when cytochrome c1 is previously reduced by ascorbate and they do not inhibit oxidation of cytochrome b by fumarate. These effects of mucidin and strobilurin A are, however, qualitatively identical with those of myxothiazol, an antibiotic that inhibits respiration by binding to cytochrome b [Von Jagow, G., Ljungdahl, P. O., Graf, P., Ohnishi, T., & Trumpower, B. L. (1984) J. Biol. Chem. 259, 6319-6326]. Mucidin and strobilurin A have identical UV and mass spectra, and they elute together on high-pressure liquid chromatography. We thus conclude that these antibiotics, although isolated from different bacteria, are structurally identical. Our results indicate that strobilurin A and mucidin inhibit electron transport at the same site as myxothiazol and not at the antimycin site, as previously reported [Subik, J., Behren, M., & Musilek, V. (1974) Biochem. Biophys. Res. Commun. 57, 17-22].     

Electronic state of heme in cytochrome oxidase III. The magnetic susceptibility of beef heart cytochrome oxidase and some of its derivatives from 7-200 K. Direct evidence for an antiferromagnetically coupled Fe (III)/Cu (II) pair.

The temperature dependence of the paramagnetic susceptibility of cytochrome oxidase and some of its derivatives has been measured from 7 to 200 K. The results obtained for the fully oxidized (resting) enzyme correspond exactly to the requirements of the model recently proposed by Palmer et al. (Palmer, G., Babcock, G. T., and Vickery, L. E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210) in which the enzyme possesses two magnetically isolated spin S = 1/2 centers and a spin-coupled S = 2 center. The S = 2 center paramagnetism has been interpreted as arising from a [cytochrome a33+(S = 5/2)--Cuu2+(S = 1/2)] antiferromagnetically coupled iron.copper binuclear complex of total spin S = 2 with -J greater than or equal to 200 cm-1. In addition, the wide temperature range used in the present studies has permitted an analysis of present and other available data (T less than 4K measurements) which readily accommodates results from this and other laboratories (Moss, T.H., Shapiro, E., King, T.E., Beinert, H., and Hartzell, C. R. (1978) J. Biol. Chem 253, 8072-8073) so that a fully consistent picture of the magnetic centers in cytochrome oxidase now appears to be available. Furthermore, anomalous magnetic behavior for the oxidized enzyme.cyanide complex has been interpreted in terms of an antiferromagnetic exchange interaction operating in the binuclear complex [cytochrome a33+.CN-(S = 1/2)--Cuu2+(S = 1/2)] with -J congruent to 40 cm-1. A structural model for the [cytochrome a3(3+)-bridge-CUu2+] center is advanced in which an imidazolate ion serves as the bridging ligand in a manner similar to that found in superoxide dismutase.     

The isolation and purification of cytochrome c1 from bovine heart

A large-scale isolation method for cytochrome c1 from beef heart is presented, based in principle on the procedure of Yu et al. (Yu, C.A., Yu, L. and King, T.E. (1972) J. Biol. Chem. 247, 1012--1019). Optimal solubilization of cytochrome c1 from succinate-cytochrome c oxidoreductase was achieved with 15% beta-mercaptoethanol, 1.5% cholate, 0.5% deoxycholate in 8% saturated ammoniun sulphate. The protein is purfied to a higher degree by chromatography on DEAE-cellulose and Ultrogel AcA 44. The method is reproducible and gives highly purified cytochrome c1 with a yield from succinate-cytochrome c oxidoreductase of 40%. The purified cytochrome c1 contains 32 nmol of heme/mg protein and has a spectral heme-to-protein ratio (Ared417nm/Ax276nm) of 2.7. Reduced cytochrome c1 is oxidized very rapidly by ferricytochrome c (k = 3 . 10(7) M-1 . S-1 at 10 degrees C, 100 mM potassium phosphate (pH 7.0) and 1% Tween 20). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate shows that the isolated protein consists of one peptide, with a molecular weight of 31 000, carrying the chromophore. In the presence of 1% sodium cholate or 1% Tween 80, cytochrome c1 is in the monomeric state, whereas at lower concentrations of detergent the protein aggregates. The aggregation of cytochrome c1 is found to be reversible.     

Photoreduction of cytochrome c1.

1. Ferricytochrome c1 solution was reduced completely between pH 7 and 10 by illumination under anaerobic conditions. Photoreduction was not affected by the ionic strength of the medium. However, it did not take place at pH lower than 6 or higher than 10, or in the presence of p-hydroxymercuric benzoate. The ferricyanide-reoxidized photoreduced c1 was not further reduced upon illumination. The reductant was most probably a specific sulfhydryl group in the subunit containing the heme of the cytochrome since this subunit contained one less p-HMB-titratable group in the photoreduced sample than in the untreated preparation. 2. The photoreduced cytochrome c1 showed the same spectra as the native cytochrome, and was not reactive with carbon monoxide. The equilibrium constant of the reaction c12+ + c3+ equilibrium c13+ + c2+ for the photoreduced c1 was found to be slightly lower (Keq = 2.6) than that for the native c1 (Keq = 3.5). The antimycin A-sensitive electron acceptor activity of ferricyanide-reoxidized photoreduced c13+ catalyzed by succinate-cytochrome c reductase was about 80% of that of the native c1. 3. A somewhat simplified method for isolation of cytochrome c1 was developed. Anaerobic ammonium sulfate fractionation and calcium phosphate gel chromatography were still used in order to achieve the purity level of about 25 nmol of heme/mg of protein. The cytochrome c1 prepared by this procedure showed the same properties tested as that by the beta-mercaptoethanol method (Yu, C.A., Yu, L., and King, T.E. (1972) J. Biol. Chem. 247, 1012-1019).     

Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase

The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of electron-transfer and proton-translocation activities in bovine heart mitochondrial cytochrome c oxidase deficient in subunit III

The electron-transfer and proton-translocation activities of cytochrome c oxidase deficient in subunit III (Mr 29 884) prepared by native gel electrophoresis [Ludwig, B., Downer, N. W., & Capaldi, R. A. (1979) Biochemistry 18, 1401-1407] have been investigated. This preparation has been depleted of 82-87% of its subunit III content as quantitated by Coomassie Brilliant Blue staining intensity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and [14C]dicyclohexylcarbodiimide labeling. The maximum rate of electron transfer of the subunit III deficient enzyme at pH 6.5 is 383 s-1, 78% of control enzyme. Neither the high-affinity site (Km = 10(-8) M) nor the low-affinity site (Km = 10(-6) M) of the cytochrome c kinetic interaction with cytochrome c oxidase is affected by the removal of subunit III. Subunit III deficient cytochrome c oxidase retains the ability to bind cytochrome c in both the high- and low-affinity sites as determined in direct thermodynamic binding experiments. Liposomes containing this preparation exhibit a respiratory control ratio [Hinkle, P. C., Kim, J. J., & Racker, E. (1972) J. Biol. Chem. 247, 1338-1341] of 3.9, while liposomes containing control enzyme exhibit a ratio of 4.3, suggesting that they have a similar proton permeability. Vectorial proton translocation initiated by the addition of ferrocytochrome c in liposomes containing subunit III deficient enzyme is decreased by 64% compared to those containing control enzyme. When the proton-translocated to electron-transferred ratio is measured in these phospholipid vesicles at constant enzyme turnover, removal of subunit III from the enzyme decreases the ratio from 0.52 to 0.21, a 60% decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.     

Sulfite oxidase from chicken liver. The role of imidazole and carboxyl groups for the reaction with cytochrome c

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor. Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomitant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite-oxidase--cytochrome-c complex. Blocking of carboxyl groups with water-soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity. The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., B?nziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683-5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191-5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron-transfer domain around the exposed heme edge.     

Gene structure of cytochrome P-450(M-1) specifically expressed in male rat liver

Cytochrome P-450(M-1) [P-450(M-1)] is specifically expressed in adult male rat liver [Yoshioka, H., Morohashi, K., Sogawa, K., Miyata, T., Kawajiri, K., Hirose, T., Inayama, S., Fujii-Kuriyama, Y., & Omura, T. (1987) J. Biol. Chem. 262, 1706-1711]. Isolation and analysis of the gene for P-450(M-1) revealed that the coding region of the gene is interrupted by eight introns and is dispersed over a 35-kilobase pair region of chromosomal DNA. Intron insertion sites of the P-450(M-1) gene are located at equivalent positions to those of cytochrome P-450b and P-450e, which are phenobarbital-inducible. Sequence analysis of the 5'-upstream region of the P-450(M-1) gene shows that there is a homologous sequence to glucocorticoid regulatory elements (GRE) identified in glucocorticoid-responsive genes.     

Cation-induced stabilization of the engineered cation-binding loop in cytochrome c peroxidase (CcP)

We have previously shown that the K(+) site found in the proximal heme pocket of ascorbate peroxidase (APX) could be successfully engineered into the closely homologous cytochrome c peroxidase (CcP) [Bonagura et al., (1996) Biochemistry 35, 6107-6115; Bonagura et al. (1999) Biochemistry 38, 5538-5545]. In addition, specificity could be switched to binding Ca(2+) as found in other peroxidases [Bonagura et al. (1999) J. Biol. Chem. 274, 37827-37833]. The introduction of a proximal cation-binding site also promotes conversion of the Trp191 containing cation-binding loop from a "closed" to an "open" conformer. In the present study we have changed a crucial hinge residue of the cation-binding loop, Asn195, to Pro which stabilizes the loop, albeit, only in the presence of bound K(+). The crystal structure of this mutant, N195PK2, has been refined to 1.9 A. As predicted, introduction of this crucial hinge residue stabilizes the cation-binding loop in the presence of the bound K(+). As in earlier work, the characteristic EPR signal of Trp191 cation radical becomes progressively weaker with increasing [K(+)] and the lifetime of the Trp191 radical also has been considerably shortened in this mutant. This mutant CcP exhibits reduced enzyme activity, which could be titrated to lower levels with increasing [K(+)] when horse heart cytochrome c is the substrate. However, with yeast cytochrome c as the substrate, the mutant was as active as wild-type at low ionic strength, but 40-fold lower at high ionic strength. We attribute this difference to a change in the rate-limiting step as a function of ionic strength when yeast cytochrome c is the substrate.