Mitochondrial Membrane-Bound Steroid Hydroxylating Systems: Structure and Functions

Ахрем, А. А.; Lapko, Veniamin N.; Лапко, А. Г.; Martsev, S. P.; Shkumatov, V. M.; Chashchin, Vadim L.

doi:10.1016/b978-0-08-023967-5.50021-8

Cited by 14 publications

(23 citation statements)

References 3 publications

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“…Both the resonances due to valine and isoleucine disappear on truncation of porcine adrenodoxin with trypsin (data not shown), suggesting that the primary sequence of porcine adrenodoxin is longer than that reported by amino acid sequencing [42]. As mentioned later, the 1 D-NMR spectrum of the porcine protein does not exhibit the resonances due to the &-methyl groups of Met120 and Met122 (see Fig.…”

Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 81%

“…More than one cross-peak to the AMX spin system of serines is missing in the spectrum for porcine adrenodoxin. Porcine adrenodoxin has been reported to be composed of 11 7 amino acids by amino acid sequencing [42] and is 11 amino acids shorter than the bovine protein so that the serines at the COOH-terminal region, Ser124 and Ser125, as well as Serll2 might be missing. Ile25 of bovine adrenodoxin is substituted by Val in the porcine protein and the corresponding spectral changes are identified in DQF-COSY spectrum (Fig.…”

Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 99%

“…5. Comparison of the primary structures of the bovine and porcine adrenodoxins shows eight non-aromatic amino acid substitutions [42], namely : Glul7+Lys, Lys22+Gln, Ile25-tVa1, Va134+Ile, Gln35-t Glu, Gln61 +Asp, Aspl09+Glu and Serl12+Ala. Consistent with this, no difference in the aromatic region of the DQF-COSY spectrum was found.…”

Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 99%

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Proton nuclear magnetic resonance investigation of adrenodoxin

Miura

Ichikawa

1991

European Journal of Biochemistry

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Bovine, porcine and sheep adrenodoxin, and the trypsin-resistant form of bovine adrenodoxin have been studied by one-and two-dimensional 'H-NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted a-protons of His10 and Phell suggest possible involvement in /I-sheet structure. The effects on the assigned resonances due to the specific spin-label with a nitroxide radical at Cys95 have been analyzed on a two-dimensional 'H-NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X-ray crystallographic data is available. &-Methyl groups of Met120 and Met122 have been assigned by comparing 'H-NMR spectra of adrenodoxin with those of the trypsin-resistant form of adrenodoxin which is specifically cleaved at Argll5. EMethyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH-terminal peptide spanning over 13 amino acids rotates rather freely in the solvent.Adrenal ferredoxin (adrenodoxin) is a small iron-sulfur protein with relative molecular mass of about 14000, which is located in the matrix of the adrenal cortex mitochondria. Adrenodoxin is synthesized with a signal peptide yielding a precursor protein [I, 21. The mature form, after being processed and translocated into mitochondria, comprises 128 amino acids based on cDNA [3] and amino acid sequencing [4, 51. It functions as an electron transfer protein from NADPHadrenodoxin reductase to cytochromes P-45OsCc (cholesterol side-chain cleavage enzyme) and P-4501 1 B (steroid 11B monooxygenase) in the steroidogenic electron transfer system [6] (and references therein).Similar iron-sulfur proteins with a 2Fe-2S cluster are distributed in a wide range of biological electron-transfer chains including plant and blue green algae [7]. Meyer et al. [8] have divided them into three classes based on the amino acid sequence similarity. Adrenodoxin and putidaredoxin from Pseudomonas putida [9], both participating in hydroxylation reactions, belong to one class. The plant ferredoxins, exemplified by ferredoxins from Spinacea oleracea (spinach) and Correspondence to S. Miura, Kagawa Medical School, Department of Biochemistry, Miki-cho, Kita-gun, Kagawa 761-07, JapanAbbreviations. COSY, J-correlated spectroscopy; DQF, double quantum filtered; NOESY, nuclear Overhauser effect spectrosocopy; HOHAHA, homonuclear Hartmann-Hahn; DANTE, delays alternating with nutations for tailored excitation; CPMG, Carr-PurcellMeilboom-Gill; 4-maleimide TEMP, 4-maleimide-2,2,6,6-tetramethyl-1 -piperidinyloxy.Enzymes. NADPH -adrenodoxin reductase (EC 1.1 8.1.2); cytochrome P-45OsCc cholesterol side-chain cleavage enzyme (EC 1.14.15.6); c...

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Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 81%

Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 99%

Section: Comparison Of Bovine With Porcine Adrenodoxinmentioning

confidence: 99%

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Proton nuclear magnetic resonance investigation of adrenodoxin

Miura

Ichikawa

1991

European Journal of Biochemistry

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“…Whereas the spin-state thermodynamics of bacterial camphor monooxygenase (P450,,,) and liver microsomal P450 are well documented (a temperature increase induces a shift from low to high spin, and the AH values depend on local pH and ionic strength) [4, 9, 101, cholesterol-side-chain-cleaving P450 (P450,,,) shows a much more confusing picture: contrarily to other P450 species, its spin state shifts from high to low spin when the temperature is increased [ll]. However, within its natural mitochondrial environment, the inverse transition takes places, as reported by Akhrem et al [12]. In view of the biological importance of P450,,, (the cholesterol-side-chaincleavage is an obligatory step in the steroid hormone biosynthesis), and also because this enzyme is often used as a model for other P450 species, we investigated further the mechanism of the P450,,, spin-state transition.…”

mentioning

confidence: 92%

“…However, within its natural mitochondrial environment, the inverse transition takes places, as reported by Akhrem et al [12]. In view of the biological importance of P450,,, (the cholesterol-side-chaincleavage is an obligatory step in the steroid hormone biosynthesis), and also because this enzyme is often used as a model for other P450 species, we investigated further the mechanism of the P450,,, spin-state transition.…”

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confidence: 99%

The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium

Lange

Larroque

Anzenbacher

1992

European Journal of Biochemistry

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We have investigated the spin-state equilibrium of adrenal mitochondrial P450,,, (cholesterolside-chain-cleaving, CYPl1 Al) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 "C to -20 "C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are AH, = 0.7 kJ . mol-' andat low temperatures and AH, = -42 kJ.mol-' and AS, = -152 J . K-' . mol-' at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH.The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.The cytochrome P450 (P450) heme-iron spin state has intrigued researchers for many years [l]. Upon substrate binding, P450 converts in most cases from a low-spin ( S = 1/2) to a high-spin ( S = 5/2) state. The spin state is coupled to the P450 redox potential: P450 reduction is more easy in the highspin than in the low-spin state [2,3], suggesting a link between spin state and enzyme function. Both, substrate-free and substrate-bound P450 are reported to exist in a thermal spin-state equilibrium [ l , 2, 41. In fact, most spin investigations are indirect : they deal with the spectrally determined equilibrium between P450 species absorbing at 417 nm and 391 nm. However, the position of the absorbance maximum in the Soret band of the ferric protein is related to the spin state of its iron center, as measured by resonance techniques such as EPR and Mossbauer spectroscopy [2, 4, 51. During a low-spin to highspin transition, the heme iron looses its sixth ligand and becomes displaced 45 pm out of the heme plane [6]. This heme structural change depends however on the ligand field produced by the surrounding protein and by the bound substrate. It is influenced by a wide spectrum of physiological and Abbreviations. P450,,,, cholesterol-side-chain-cleaving cytochrome P450; P450,,,, camphor monooxygenase.Enzymes. Cytochrome P450,,,, cholesterol monooxygenase (sidechain-cleaving) (EC 1.14.15.6); cytochrome P450,,,, camphor monooxygenase (EC 1.14.15.1).Mende (CNRS), F-34033 Montpellier, France physicochemical parameters [7]. The spin state is therefore a potentially helpful parameter for the detection of proteinconformational changes. Especially, the thermodynamic parameters AH and AS of this reaction have been used to unravel discrete protein-conformational changes [4, 8, 91. Whereas the spin-state thermodynamics of ba...

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Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

2001

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The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.

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Mitochondrial Membrane-Bound Steroid Hydroxylating Systems: Structure and Functions

Cited by 14 publications

References 3 publications

Proton nuclear magnetic resonance investigation of adrenodoxin

Proton nuclear magnetic resonance investigation of adrenodoxin

The cholesterol‐side‐chain‐cleaving cytochrome P450 spin‐state equilibrium

Functional Display of Active Bovine Adrenodoxin on the Surface of E. coli by Chemical Incorporation of the [2Fe–2S] Cluster

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