Nitrogenous ligation at the sixth coordination position of the Thr-301 to Lys-mutated P450IIC2 heme iron.

Threonine-301 of P450IIC2 was replaced by lysine via site-directed mutagenesis. The Lys-mutated P450 exhibited absorption spectra that were characteristic of the nitrogenous-ligand-bound form of P450. In the oxidized form, the Soret band was red-shifted as compared with the typical ferric low-spin form of P450 and the beta band was more intense than the alpha band. In the reduced form, two Soret peaks were observable at 447 and 423 nm and their relative heights were dependent on pH, indicating the existence of two interconvertible states of ferrous Lys-mutated P450 which are in equilibrium. In addition, the interaction of external ligands with the P450 heme iron was profoundly inhibited both in the oxidized and reduced forms. These findings suggest that epsilon-amino nitrogen of Lys-301, which was introduced by amino acid substitution, occupies the 6th coordination position with the heme iron of the Lys-mutated P450, because, owing to conformation of the P450 protein, the epsilon-amino group may be located at just the right position for coordination as the internal 6th ligand.     

Biophysical characterization of the sterol demethylase P450 from Mycobacterium tuberculosis, its cognate ferredoxin, and their interactions

Mycobacterium tuberculosis encodes a P450 of the sterol demethylase family (CYP51) chromosomally located adjacent to a ferredoxin (Fdx). CYP51 and Fdx were purified to homogeneity and characterized. Spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron in CYP51. An epsilon419 of 134 mM(-1) cm(-1) was determined for oxidized CYP51. Analysis of interactions of 1-, 2-, and 4-phenylimidazoles with CYP51 showed that the 1- and 4-forms were heme iron-coordinating inhibitors, while 2-phenylimidazole induced a substrate-like optical shift. The 2-phenyimidazole-bound CYP51 demonstrated unusual decreases in high-spin heme iron content at elevated temperatures and an almost complete absence of high-spin heme iron by low-temperature EPR. These data suggest thermally induced alterations in CYP51 active site structure and/or binding modes for the small ligand. Reduction of CYP51 in the presence of carbon monoxide leads to formation of an Fe(II)-CO complex with a Soret absorption maximum at 448.5 nm, which collapses (at 0.246 min(-1) at pH 7.0) forming a species with a Soret maximum at 421.5 nm (the inactive P420 form). The rate of P420 formation is accelerated at lower pH, consistent with protonation of the cysteinate (Cys 394) to a thiol underlying the P450-P420 transition. The P450 form is stabilized by estriol, which induces a type I spectral shift on binding CYP51 (Kd = 21.7 microM). Nonstandard spectral changes occur on CYP51 reduction (using either dithionite or natural redox partners), including a blue-shifted Soret band and development of a strong feature at approximately 558.5 nm, suggestive of cysteine thiol ligation. Thus, ligand-free ferrous CYP51 is prone to thiolate ligand protonation even in the absence of carbon monoxide. Analysis of reoxidized CYP51 demonstrates that the enzyme re-forms P450, indicating that Cys 394 thiol is readily deprotonated to thiolate in the ferric form. Spectroscopic analysis of Fdx by EPR (resonance at g = 2.03) and magnetic CD (intensity for oxidized and reduced forms and signal intensity dependence on field strength and temperature) demonstrated that Fdx binds a [3Fe-4S] iron-sulfur cluster. Potentiometric studies show that the midpoint potential for ligand-free CYP51 is -375 mV, increasing to -225 mV in the estriol-bound form. The Fdx potential is -31 mV. Fdx forms a productive electron transfer complex with CYP51 and reduces it at a rate of 3.0 min(-1) in the ligand-free form and 4.3 min(-1) in the estriol-bound form, despite a thermodynamic barrier. Steady-state analysis of a M. tuberculosis class I redox system comprising flavoprotein reductase A (FprA), Fdx, and estriol-bound CYP51 indicates heme iron reduction as a rate-limiting step.     

Characterization of a dissimilatory-type sulfite reductase, desulfoviridin, from Desulfovibrio africanus Benghazi

A desulfoviridin-type sulfite reductase having the alpha band at 638 nm was purified from Desulfovibrio africanus Benghazi (NCIB 8401) by chromatography on DEAE-cellulose, Sephadex G-200, and DEAE-Sepharose columns and by disc gel electrophoresis. The content of desulfoviridin in the soluble protein was estimated to be about 6% from the purification indexes. Like the typical desulfoviridin from D. vulgaris Miyazaki K, it formed mainly trithionate besides thiosulfate and sulfide in sulfite reduction coupled to hydrogenase and methyl viologen. No significant differences in the amino acid compositions, CD patterns in the UV (205-250 nm) region, and subunit structures were found, except for a pI value about 1 unit larger (pI 5.3). The split Soret (410 +/- 2 nm, less intense peak at 391 +/- 2 nm with a shoulder around 380 nm) and beta (584 +/- 2 nm) band maxima of the enzyme as isolated, and the visible absorption and fluorescence spectra of the acidic acetone-extracted chromophore were almost identical to those ascribed to sirohydrochlorin in spite of the reported difference in the native enzyme (alpha band maxima at 638 nm as against 628 +/- 2 nm in a typical desulfoviridin). Iron was the only significant chelatable metal contained in the chromophore. Some differences between africanus and vulgaris desulfoviridins were observed in the CD patterns in the UV to near UV region (250-340 nm) and also in the visible absorption spectra in the presence of dithionite.     

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

Rapid P450 heme iron reduction by laser photoexcitation of Mycobacterium tuberculosis CYP121 and CYP51B1. Analysis of CO complexation reactions and reversibility of the P450/P420 equilibrium

We demonstrate that photoexcitation of NAD(P)H reduces heme iron of Mycobacterium tuberculosis P450s CYP121 and CYP51B1 on the microsecond time scale. Rates of formation for the ferrous-carbonmonoxy (Fe(II)-CO) complex were determined across a range of coenzyme/CO concentrations. CYP121 reaction transients were biphasic. A hyperbolic dependence on CO concentration was observed, consistent with the presence of a CO binding site in ferric CYP121. CYP51B1 absorption transients for Fe(II)-CO complex formation were monophasic. The reaction rate was second order with respect to [CO], suggesting the absence of a CO-binding site in ferric CYP51B1. In the absence of CO, heme iron reduction by photoexcited NAD(P)H is fast ( approximately 10,000-11,000 s(-1)) with both P450s. For CYP121, transients revealed initial production of the thiolate-coordinated (P450) complex (absorbance maximum at 448 nm), followed by a slower phase reporting partial conversion to the thiol-coordinated P420 species (at 420 nm). The slow phase amplitude increased at lower pH values, consistent with heme cysteinate protonation underlying the transition. Thus, CO binding occurs to the thiolate-coordinated ferrous form prior to cysteinate protonation. For CYP51B1, slow conversions of both the ferrous/Fe(II)-CO forms to species with spectral maxima at 423/421.5 nm occurred following photoexcitation in the absence/presence of CO. This reflected conversion from ferrous thiolate- to thiol-coordinated forms in both cases, indicating instability of the thiolate-coordinated ferrous CYP51B1. CYP121 Fe(II)-CO complex pH titrations revealed reversible spectral transitions between P450 and P420 forms. Our data provide strong evidence for P420 formation linked to reversible heme thiolate protonation, and demonstrate key differences in heme chemistry and CO binding for CYP121 and CYP51B1.     

Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set

A conserved glutamate covalently attaches the heme to the protein backbone of eukaryotic CYP4 P450 enzymes. In the related Bacillus megaterium P450 BM3, the corresponding residue is Ala264. The A264E mutant was generated and characterized by kinetic and spectroscopic methods. A264E has an altered absorption spectrum compared with the wild-type enzyme (Soret maximum at approximately 420.5 nm). Fatty acid substrates produced an inhibitor-like spectral change, with the Soret band shifting to 426 nm. Optical titrations with long-chain fatty acids indicated higher affinity for A264E over the wild-type enzyme. The heme iron midpoint reduction potential in substrate-free A264E is more positive than that in wild-type P450 BM3 and was not changed upon substrate binding. EPR, resonance Raman, and magnetic CD spectroscopies indicated that A264E remains in the low-spin state upon substrate binding, unlike wild-type P450 BM3. EPR spectroscopy showed two major species in substrate-free A264E. The first has normal Cys-aqua iron ligation. The second resembles formate-ligated P450cam. Saturation with fatty acid increased the population of the latter species, suggesting that substrate forces on the glutamate to promote a Cys-Glu ligand set, present in lower amounts in the substrate-free enzyme. A novel charge-transfer transition in the near-infrared magnetic CD spectrum provides a spectroscopic signature characteristic of the new A264E heme iron ligation state. A264E retains oxygenase activity, despite glutamate coordination of the iron, indicating that structural rearrangements occur following heme iron reduction to allow dioxygen binding. Glutamate coordination of the heme iron is confirmed by structural studies of the A264E mutant (Joyce, M. G., Girvan, H. M., Munro, A. W., and Leys, D. (2004) J. Biol. Chem. 279, 23287-23293).     

Structural characterization of n-butyl-isocyanide complexes of cytochromes P450nor and P450cam

Alkyl-isocyanides are able to bind to both ferric and ferrous iron of the heme in cytochrome P450, and the resulting complexes exhibit characteristic optical absorption spectra. While the ferric complex gives a single Soret band at 430 nm, the ferrous complex shows double Soret bands at 430 and 450 nm. The ratio of intensities of the double Soret bands in the ferrous isocyanide complex of P450 varies, as a function of pH, ionic strength, and the origin of the enzyme. To understand the structural origin of these characteristic spectral features, we examined the crystallographic and spectrophotometric properties of the isocyanide complexes of Pseudomonas putida cytochrome P450cam and Fusarium oxysporum cytochorme P450nor, since ferrous isocyanide complex of P450cam gives a single Soret band at 453 nm, while that of P450nor gives one at 427 nm. Corresponding to the optical spectra, we observed C-N stretching of a ferrous iron-bound isocyanide at 2145 and 2116 cm(-1) for P450nor and P450cam, respectively. The crystal structures of the ferric and ferrous n-butyl isocyanide complexes of P450cam and P450nor were determined. The coordination structure of the fifth Cys thiolate was indistinguishable for the two P450s, but the coordination geometry of the isocyanide was different for the case of P450cam [d(Fe-C) = 1.86 A, angleFe-C-N = 159 degrees ] versus P450nor [d(Fe-C) = 1.85 A, angleFe-C-N = 175 degrees ]. Another difference in the structures was the chemical environment of the heme pocket. In the case of P450cam, the iron-bound isocyanide is surrounded by some hydrophobic side chains, while, for P450nor, it is surrounded by polar groups including several water molecules. On the basis of these observations, we proposed that the steric factors and/or the polarity of the environment surrounding the iron-bound isocyanide significantly effect on the resonance structure of the heme(Fe)-isocyanide moiety and that differences in these two factors are responsible for the spectral characteristics for P450s.     

Interaction of aromatic cytokinins with human liver microsomal cytochromes P450

Aromatic cytokinins (ortho-topolin riboside, 6-benzylaminopurine riboside and 6-(2-hydroxy-3-methoxybenzyla mino)purine riboside) were tested for their possible interaction with human liver microsomal cytochromes P450 by absorption difference spectroscopy. All three compounds were shown to bind to the CYP enzymes producing a high to low spin shift of the heme iron yielding a Soret absorption band shift to approximately 425 nm. As this type of spectral change means that the substance is able to bind directly to the heme iron, the results obtained open the possibility of an interaction of these compounds with metabolism of other drugs or, in general, with other substrates of cytochromes P450.     

Absorption spectroscopy and circular dichroism studies of sperm whale myoglobin chemically modified on the N-terminal α-aminogroup by isothiocyanate reagents

Sperm whale metMb [Mb(SW)] was modified chemically by fluorescein isothiocyanate and methylisothiocyanate. Individual modification products on the α-aminogroup of the N-terminal Val were isolated with ion exchange chromatography (FITC-Mb and MITC-Mb). Absorption spectra in the 200–700 nm region and spectrophotometric titration curves in the Soret band of the modified metMb derivatives and intact metMb were compared. Characteristic differences between them indicate that upon modification there occurs a shift in the equilibrium of isomers of the metMb aquo complex towards the low-spin form. The CD spectra of FITC-metMb and MITC-inetMb in the 200–450 nm region attest to small changes in the heme environment as compared to native metMb without, however, any appreciable conformational changes of the polypeptide chain. No differences have been found in the absorption and CD spectra in the Soret region between native deoxy-Mb and the modified Mb derivatives in deoxy forms. An analysis of the present results and of those reported in the literature shows that the conformational changes at the N-end of Mb upon modification of the N-terminal α-amino group result in structural alterations in the heme environment which are most likely to consist in some reorientation of the side group of His E7 and, possibly, those of phe B14, Phe CD1,and Phe CD4 on the distal side of the heme. A scheme of the electronic conformational interactions (ECI) in ferrimyoglobin is proposed.     

鸡细胞色素P450 1A5的原核表达与活性重组

旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。     

Characterization of active site structure in CYP121. A cytochrome P450 essential for viability of Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.     

Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 from Corynebacterium sp. strain EP1

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1A showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1A had a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1A degraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+ oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1A catalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1A metabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.     

On the denaturation of porcine erythrocyte catalase with alkali, urea, and guanidine hydrochloride in relation to its subunit structure

The dissociation of porcine erythrocyte catalase [EC 1.11.1.6] into subunits on denaturation with alkali, GuHCl and urea was investigated by following the changes in hydrodynamic properties, absorption and CD spectra in the Soret region and inactivation of the enzyme. It was found that dissociation proceeded in an "all or none" manner from the native tetramer (molecular weight, ca. 250,000) into identical 1/4-sized monomers (molecular weight, ca. 54,000 with alkali, 65,000 with urea and 71,000 with GuHCl) as estimated by ultracentrifugal analyses. On this dissociation, the sedimentation coefficient decreased from about 11S to 5.1 - 3.7S, and absorption spectra in the Soret region decreased to about 40% of the native level and showed a broad band around 365-375 nm and a shoulder around 415-420 nm; these changes were accompanied by complete loss of enzyme activity. The change in enzyme activity correlated well with that of absorption and CD spectra in the Soret region, depending on denaturation time, alkaline pH used and concentration of both denaturants. The reassociated catalase obtained by removing urea by dialysis was characterized by recovery of distinct CD bands in the Soret and near ultraviolet regions, although the partial refolding of alpha-helical conformation occurred without recovery of enzyme activity. These results indicate that the conformational changes and dissociation process of catalase into subunits can be monitored spectrophotometrically in relation to enzyme activity, and that subtle conformations near the heme groups and polypeptide backbone play an important role in maintaining full enzyme activity of the catalase molecule.     

Ligand binding of organic sulfides to microsomal cytochrome P-450.

Octyl methyl-, butyl methyl- and pentamethylene sulfide react with about 50% of oxidized cytochrome P-450 in liver microsomes from phenobarbital-pretreated rats by formation of optical difference spectra with maxima at 435 and 552 nm and concomitant shifts in the electron paramagnetic resonance spectrum. Reduction by NADPH or sodium dithionite yielded a Soret absorption band at 449 nm and alpha and beta bands at 573 and 545 nm, respectively. The ligand metyrapone and the substrate n-octane competitively inhibited the formation of these difference spectra and pentamethylene sulfide was a strong competitive inhibitor of the 0-deakylation of 7-ethoxycoumarin. These results indicate a direct ligand binding of the sulfides to cytochrome P-450 with concomitant blocking of the hydrophobic substrate binding site. Some sulfides did not interact as ligands but as substrates, in variation, however, with the source of microsomes.     

Mycobacterium tuberculosis CYP130: crystal structure, biophysical characterization, and interactions with antifungal azole drugs

CYP130 is one of the 20 Mycobacterium tuberculosis cytochrome P450 enzymes, only two of which, CYP51 and CYP121, have so far been studied as individually expressed proteins. Here we characterize a third heterologously expressed M. tuberculosis cytochrome P450, CYP130, by UV-visible spectroscopy, isothermal titration calorimetry, and x-ray crystallography, including determination of the crystal structures of ligand-free and econazole-bound CYP130 at a resolution of 1.46 and 3.0A(,) respectively. Ligand-free CYP130 crystallizes in an "open" conformation as a monomer, whereas the econazole-bound form crystallizes in a "closed" conformation as a dimer. Conformational changes enabling the "open-closed" transition involve repositioning of the BC-loop and the F and G helices that envelop the inhibitor in the binding site and reshape the protein surface. Crystal structure analysis shows that the portion of the BC-loop relocates as much as 18A between the open and closed conformations. Binding of econazole to CYP130 involves a conformational change and is mediated by both a set of hydrophobic interactions with amino acid residues in the active site and coordination of the heme iron. CYP130 also binds miconazole with virtually the same binding affinity as econazole and clotrimazole and ketoconazole with somewhat lower affinities, which makes it a plausible target for this class of therapeutic drugs. Overall, binding of the azole inhibitors is a sequential two-step, entropy-driven endothermic process. Binding of econazole and clotrimazole exhibits positive cooperativity that may reflect a propensity of CYP130 to associate into a dimeric structure.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

R-phycoerythrins having two conformations for the same aggregate

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.     

Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis.

The bioI gene has been sub-cloned and over-expressed in Escherichia coli, and the protein purified to homogeneity. The protein is a cytochrome P450, as indicated by its visible spectrum (low-spin haem iron Soret band at 419 nm) and by the characteristic carbon monoxide-induced shift of the Soret band to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochrome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the protein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose). The purified protein is of mixed spin-state by both electronic spectroscopy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1.91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichroism and electron paramagnetic resonance studies indicate that P450 BioI has a cysteine-ligated b-type haem iron and the near-IR magnetic circular dichroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typical of cytochrome P450, notably the oxidation state marker v4 at 1,373 cm(-1) (indicating ferric P450 haem) and the splitting of the spin-state marker v3 into two components (1,503 cm(-1) and 1,488 cm(-1)), indicating cytochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (Kd=4.18+/-0.26 microM) and pentadecanoic acid (Kd=3.58+/-0.54 microM) having highest affinity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (Kd<1 microM), again indicating strong affinity for fatty acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin reductase. Substrate hydroxylation at the omega-terminal position was demonstrated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoic acid, and by separation of product from the reaction of P450 BioI with myristic acid.     

Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme.

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.