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H Kamin

Publications and source records attributed to H Kamin.

At least 19 recordsLinked to original sources
PubMed1986

Association of ferredoxin-NADP+ reductase with NADP(H) specificity and oxidation-reduction properties.

The equilibrium properties of the NADP+ binding site of ferredoxin-NADP+ reductase (FNR, or Fd-NADP+ reductase) were examined with regard to specificity in binding, and with regard to the oxidation-reduction properties of the FNR.NADP+ complex. With the exception of 3'-NADP+, only adenosine nucleotides with a 2'-adenosyl phosphate bound to Fd-NADP+ reductase. Kd values increased in the order: 2',5'-ADP greater than 2',5'-ATP ribose greater than NADP+ greater than 2'-AMP greater than 3'-NADP+. No evidence was found for binding of NAD, NMN, or 5'-ADP. Thus the 2'-adenosylphosphate controls specificity in substrate binding, as well as specificity in enzyme activity. The low affinity of Fd-NADP+ reductase for 2'-AMP suggests that the phosphate(s) of the pyrophosphate bridge of NADP+ may also contribute significantly to binding energy. Fd-NADP+ reductase was found to form a high-affinity two-electron reduced complex (FNR.NADPH) with a NADPH; complex formation was associated with appearance of long-wavelength charge-transfer bands. Kd of FNR.NADPH complex was about 6% the Kd of oxidized FNR.NADP+ complex. As predicted by the lower Kd, the Em for reduction of FNR.NADP+ complex to the charge-transfer complex was about 40 mV more positive than the potential of the NADP+/NADPH couple. Rapid kinetic studies supported description of the charge-transfer complex as primarily oxidized FNR.NADPH. Thus, complex formation helps drive electron transfer from the flavoprotein to NADP+.

Adenine Nucleotides↗
PubMed1985

Interactions between spinach ferredoxin-nitrite reductase and its substrates. Evidence for the specificity of ferredoxin.

Reduced ferredoxin can serve as electron donor in the 6-electron reduction of nitrite to ammonia catalyzed by spinach nitrite reductase. We have examined interactions between nitrite reductase and its substrates, ferredoxin and nitrite, with emphasis upon protein-protein interactions between ferredoxin and nitrite reductase. Ferredoxin, of the proteins tested, is the most effective in retarding low ionic strength inactivation of nitrite reductase. The interaction appears to be electrostatic, and the apparent Kd, calculated from the concentration dependence of ferredoxin protection, is about 1 microM in 2 mM Tris. Chemical modification of carboxyl residues of ferredoxin resulting in a change of charge reduces its reactivity with both ferredoxin:NADP+ oxidoreductase and nitrite reductase, indicating the importance of charge-charge interactions. Cross-linking studies provided no evidence for a ternary complex containing the oxidoreductase and nitrite reductase but indicated that the two enzymes will compete for ferredoxin, possibly using the same site (or overlapping sites) on the ferredoxin. A complex containing ferredoxin:NADP+ oxidoreductase, ferredoxin, and cytochrome c was detected, indicating that ferredoxin has different binding sites for cytochrome c and ferredoxin:NADP+ oxidoreductase. Active cross-linked complexes of ferredoxin and nitrite reductase were obtained and were less sensitive to low ionic strength inactivation than free reductase and had decreased ferredoxin-supported nitrite reductase activity. The evidence presented of protein-protein interactions between ferredoxin and nitrite reductase indicates that ferredoxin is indeed the specific physiological electron donor to the reductase.

Binding Sites↗
PubMed1985

Paramagnetic probes of multicomponent electron-transfer systems.

Electron-transfer systems associated with steroid hormone production in the adrenals and with photosynthetic NADP+ reduction in the chloroplast share some important features. Complexes between components in both systems affect the exposure of prosthetic groups to water-soluble paramagnetic groups. The sensitivity of the components to probes of different charges suggests that electrostatic interactions are important in complex formation. Haem plane orientation in cytochrome P-450 of the adrenal system is parallel to the membrane plane in both native and reconstituted systems.

Adrenal Glands↗
PubMed1984

Electron transfer by ferredoxin:NADP+ reductase. Rapid-reaction evidence for participation of a ternary complex.

Rapid reaction studies presented herein show that ferredoxin:NADP+ oxidoreductase (FNR, EC 1.18.1.2) catalyzes electron transfer from spinach ferredoxin (Fd) to NADP+ via a ternary complex, Fd X FNR X NADP+. In the absence of NADP+, reduction of ferredoxin:NADP+ reductase by Fd was much slower than the catalytic rate: 37-80 s-1 versus at least 445 e-s-1; dissociation of oxidized spinach ferredoxin (Fdox) from one-electron reduced ferredoxin:NADP+ reductase (FNRsq) limited the reduction of FNR. This confirms the steady-state kinetic analysis of Masaki et al. (Masaki, R., Yoshikaya, S., and Matsubara, H. (1982) Biochim. Biophys. Acta 700, 101-109). Occupation of the NADP+ binding site of FNR by NADP+ or by 2',5'-ADP (a nonreducible NADP+ analogue) greatly increased the rate of electron transfer from Fd to FNR, releiving inhibition by Fdox. NADP+ (and 2',5'-ADP) probably facilitate the dissociation of Fdox; equilibrium studies have shown that nucleotide binding decreases the association of Fd with FNR (Batie, C. J. (1983) Ph.D. dissertation, Duke University; Batie, C. J., and Kamin, H. (1982) in Flavins and Flavoproteins VII (Massey, V., and Williams, C. H., Jr., eds) pp. 679-683, Elsevier, New York; Batie, C.J., and Kamin, H. (1982) Fed. Proc. 41, 888; and Batie, C.J., and Kamin, H. (1984) J. Biol. Chem. 259, 8832-8839). Premixing Fd with FNR was found to inhibit the reaction of the flavoprotein with NADP+ and with NADPH; thus, substrate binding may be ordered, NADP+ first, then Fd. FNRred and NADP+ very rapidly formed an FNRred X NADP+ complex with flavin to nicotinamide charge transfer bands. The Fdred X NADP+ complex then relaxed to an equilibrium species; the spectrum indicated a predominance of FNRox X NADPH charge-transfer complex. However, charge-transfer species were not observed during turnover; thus, their participation in catalysis of electron transfer from Fd to NADP+ remains uncertain. The catalytic rate of Fd to NADP+ electron transfer, as well as the rates of electron transfer from Fd to FNR, and from FNR to NADP+ were decreased when the reactants were in D2O; diaphorase activity was unaffected by solvent. On the basis of the data presented, a scheme for the catalytic mechanism of catalysis by FNR is presented.

Adenosine Diphosphate↗
PubMed1984

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin.

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.

Binding Sites↗
PubMed1983

Kinetics of O2 and CO Binding to adrenal cytochrome P-450scc. Effect of cholesterol, intermediates, and phosphatidylcholine vesicles.

The kinetics of O2 and CO binding to purified adrenal cytochrome P-450scc has been measured at 25 degrees C by stopped flow spectrophotometry. Measurements were made on the cytochrome in both aqueous buffer and phosphatidylcholine vesicles; little difference in the kinetic constants between the two was observed. 22R-Hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol, intermediates in the cytochrome P-450scc-catalyzed conversion of cholesterol to pregnenolone, markedly influenced rates and equilibria of O2 and CO binding. Binding of the intermediates greatly decreased the association rate of both O2 (80-160-fold) and CO (30-80-fold) relative to cholesterol-bound cytochrome. They had opposite effects on O2 and CO dissociation rates, causing a 5-fold increase in the CO dissociation rate relative to cholesterol and a 150-900-fold decrease in the O2 dissociation rate. Kd values for CO, determined by titration of the reduced cytochrome with CO, increased from 0.3 microM for substrate-depleted cytochrome to 0.7-1.0 microM, 100 microM, and 213-244 microM, respectively, for cholesterol, 22R-hydroxycholesterol, and 20 alpha,22R-dihydroxycholesterol. Conversely, Kd values for O2, determined from the ratio of dissociation and association rate constants, decreased from 23 microM for cholesterol-bound cytochrome to 12 microM and 5 microM for 22R-hydroxycholesterol and 20 alpha,22R-hydroxycholesterol, respectively, indicating a thermodynamic stabilization of the Fe2+ . O2 complex by these intermediates.

Adrenal Cortex↗
PubMed1982

The oxyferro complex of adrenal cytochrome P-450scc. Effect of cholesterol and intermediates on its stability and optical characteristics.

The binding of O2 to reduced cytochrome P-450 is the initial step in the activation of oxygen; subsequent addition of a second electron leads to substrate hydroxylation. Without the second electron, the complex between reduced cytochrome and O2 (oxyferro) undergoes internal electron transfer to regenerate the oxidized enzyme and, presumably, superoxide. We have used 38% ethylene glycol and subzero temperatures to stabilize the oxyferro complex of cytochrome P-450scc and have examined the effect of cholesterol, and hydroxycholesterol intermediates in the conversion of cholesterol to pregnenolone, on the complex. The binding of cholesterol or the intermediates 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol, and 20 alpha, 22R-dihydroxycholesterol to the cytochrome perturbed the optical spectra of the oxyferro complex with Soret maximums varying from 416 to 423 nm. Activation energies for the autooxidation of each of these sterol-oxyferro complexes were similar (approximately 22 kcal/mol). The half-time for autooxidation of the oxyferro complex was increased 15-fold by cholesterol over substrate-free cytochrome, and the hydroxycholesterols caused a further 3-17-fold increase in the stability of the oxyferro complex over that observed for cholesterol, the stability increasing with the number of hydroxyl groups on the cholesterol side chain. This was observed in both 38% ethylene glyco at -17 degrees C and dioleoyl phosphatidylcholine vesicles at 2 degrees C. The data indicate that the 1-electron-reduced-oxygenated complex of cytochrome P-450scc is kinetically stabilized by the binding of the reaction intermediates, preserving the complex for the arrival of the second electron.

Adrenal Cortex↗
PubMed1982

Steroidogenic electron transport in adrenal cortex mitochondria.

The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxin-adrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospholipid binds.

Adrenal Cortex↗
PubMed1981

The relation of pH and oxidation-reduction potential to the association state of the ferredoxin . ferredoxin:NADP+ reductase complex.

The interaction between spinach ferredoxin and ferredoxin:NADP+ reductase was studied by varying pH and oxidation-reduction state. The Kd of the oxidized ferredoxin . ferredoxin:NADP+ reductase complex increases with increasing pH in the range from pH 6 to pH 8; the Kd is pH-independent above pH 9. These data are interpreted as showing 1 proton binding/complex at neutral pH. The extent of association of the complex was also varied by manipulation of salt concentration, was also varied by manipulation of salt concentration, and of concentration of the individual proteins. Increased association accompanied a negative shift in potential of ferredoxin relative to that of ferredoxin: NADP+ reductase. Oxidation-reduction titrations showed that the oxidation-reduction potential of ferredoxin is reduced to above -510 mV when in complex with ferredoxin:NADP+ reductase, a change of about -90 mV; the potential of ferredoxin:NADP+ reductase is changed little (no more than +20 mV). Conversely, these data also showed that the oxidation-reduction state of ferredoxin strongly affected its association with the flavoprotein, increasing the Kd at least 30-fold on reduction of ferredoxin.

Ferredoxin-NADP Reductase↗
PubMed1980

Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.

Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.

Adrenal Cortex↗
PubMed1979

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.

Adrenal Cortex↗
PubMed1979

Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.

We have shown (Seybert, D., Lambeth, D., and Kamin, H. (1978), J. Biol. Chem. 253, 8355-8358) that, whereas the 1:1 complex between adrenodoxin reductase and adrenodoxin is the active species for cytochrome c reduction, the complex is not sufficient to allow cytochrome P-45011 beta-mediated hydroxylations;adrenodoxin in excess of reductase is required. In the present studies, reduction by NADPH of excess adrenodoxin is shown to occur at a rate sufficient to support both cytochrome P-450 11 beta-mediated hydroxylation of deoxycorticosterone, and cytochrome P-450sec-mediated side chain cleavage of cholesterol. Oxidation-reduction potential and ion effect studies indicate that the mechanism of steroidogenic electron transport involves an adrenodoxin electron "shuttle" rather than a macromolecular complex of reductase, adrenodoxin, and cytochrome. The oxidation-reduction potential of adrenodoxin is shifted about -100 mV when bound to reductase, and reduction of the iron-sulfur protein thus promotes dissociation of the complex. The rate of adrenodoxin reduction is first stimulated, then inhibited by increasing salt; the effect is ion-specific, with Ca2+ approximately Mg2+ greater than Na+ greater than NH/+. Similar ion-specific rate effects are observed for both of the cytochrome P-450-mediated hydroxylations, indicating that the same reduction mechanism is required for these reactions. Increasing salt concentrations caused dissociation of the complex; dissociation of the form of the complex containing reduced adrenodoxin occurred at lower salt concentrations than that containing oxidized adrenodoxin. The order of effectiveness of ions in causing dissociation is the same as the order for stimulation of adrenodoxin reduction, suggesting a dissociation step in the mechanism. This proposed model, together with dissociation constants for the form of the complex containing either oxidized or reduced adrenodoxin, allows accurate prediction of the salt rate effects curve. For all ions, an activity maximum is seen at the ion concentration which produces the largest molar difference between associated-oxidized and dissociated-reduced states, and the model predicts the positions of the maxima for adrenodoxin reduction, 11 beta-hydroxylation, and side chain cleavage. Thus reduction-induced dissociation of adrenodoxin from adrenodoxin reductase appears to be a required step in steroidogenic electron transport by this system, and a role for adrenodoxin as a mobile electron shuttle is proposed.

Adrenal Cortex↗