[15] Purification of bacterial cytochrome P-450

O'Keeffe, David H.; Ebel, Richard E.; Peterson, Julian A.

doi:10.1016/s0076-6879(78)52017-x

Cited by 76 publications

(46 citation statements)

References 4 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…P-450soy exists as a single polypeptide with a molecular weight of 47,500. This is within the range (45,000 to 57,000) described for the majority of cytochromes P-450 (1,2,4,21,22,32). The spectral characteristics of the P-450so, are also similar to those of the other purified cytochromes P-450 (4) Cytochrome P-450S.Y is therefore the first substrate-free high-spin procaryotic P-450 to be reported, ranking among the limited number of high-spin cytochromes P-450, all isolated from either rat liver (24,31) or rabbit liver (5,13) sources.…”

Section: Discussionsupporting

confidence: 76%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

View full text Add to dashboard Cite

A soybean flour-induced, soluble cytochrome P-450 (P-450,,.Y) was purified 130-fold to homogeneity from Streptomyces gyiseus. Native cytochrome P-450,,. is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450S exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450O had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin;NADP+ oxidoreductase, purified cytochrome P-450O, catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450SOYA.Streptomyces griseus (ATCC 13273) has the remarkable ability to catalyze the stereo-and regiospecific oxidation of a diverse array of xenobiotics. The types of reactions performed by S. griseus include aromatic, cyclic, and aliphatic hydroxylations, 0-, S-, and N-oxidations, C-C fission, epoxidation, and 0-and N-dealkylations. These transformations, which occur with compounds such as alkaloids (17,27), coumarins (25), rotenoids (28), chromenes (30), and other complex xenobiotics (7,26), parallel those performed by mammalian cytochromes P-450 (34, 36). However, the nature of the enzymatic system(s) employed by S. griseus for such diverse reactions has remained unresolved (26). The biotransformations catalyzed by S. griseus occur primarily following growth of the organism on a complex medium enriched with soybean flour (26). Recently, Sariaslani and Kunz (29) demonstrated that soybean flour and one of its isoflavonoid constituents, genistein, induce the synthesis of cytochrome P-450 in S. griseus and inferred a role for this enzyme in the biotransformation reactions performed by this organism. Here we report the purification and characterization of a soybean flour-induced cytochrome P-450 and demonstrate the transformation of two xenobiotic substrates, precocene II and 7-ethoxycoumarin, by purified preparations of this enzyme.( was determined by the method of Omura and Sato (23). Heme was analyzed as its reduced pyridine hemochrome derivative (10). High-pressure liquid chromatography (HPLC) was performed on a system consisting of an LKB 2152 controller, LKI3 2150 pump, and a Hewlett-Packard 1040A diode array detector.Cytochrome P-450.,y purification protocol. All procedures except steps 4 and 5 were carried out at 4°C. Glycerol (20% [vol/vol]) was included in all buffers following the salt precipitation step to prevent degradation of the P-450 to its inactive P-420 species. Protease inhibitors used in steps 1 to 3 to inhibit a wide range of protease activities were pepstatin (an acid protease inhibitor), leupeptin (a broad-range protease inhibitor), and phenylmethylsulfonyl fluoride (PMSF; a serine protease inhibitor). Fractions were stored between purification steps at -80°C with no discernable loss o...

show abstract

Section: Discussionsupporting

confidence: 76%

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

1989

View full text Add to dashboard Cite

show abstract

“…This indicates that NOS can undergo reversible complex formation with NO during catalysis. NOS is therefore similar to certain cytochrome P450s that generate stable ferrous-nitrosyl complexes (27,30) and is dissimilar to others whose ferrousnitrosyl complexes rapidly form catalytically inactive cytochrome P420 (26). The breakdown of ferrous-nitrosyl NOS observed in our study was considerably faster than the ferrousnitrosyl complexes of hemoglobin and myoglobin under similar conditions.…”

Section: Decay Of the Ferrous-nitrosyl Complexsupporting

confidence: 48%

Neuronal Nitric Oxide Synthase Self-inactivates by Forming a Ferrous-Nitrosyl Complex during Aerobic Catalysis

Abu-Soûd

Wang

Rousseau

et al. 1995

Journal of Biological Chemistry

203

219

View full text Add to dashboard Cite

Neuronal NO synthase (NOS) is a flavin-containing hemeprotein that generates NO from L-arginine, NADPH, and O 2 . NO has recently been proposed to autoinhibit NOS. We have investigated whether a NOS heme-NO complex forms during aerobic steady-state catalysis. Visible and resonance Raman spectra recorded during steady-state NO synthesis by NOS showed that the majority of enzyme (70 -90%) was present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O 2 . Its level remained constant during NO synthesis until the NADPH was exhausted, after which the complex decayed to regenerate ferric resting NOS. Stoppedflow measurements revealed that the buildup of the ferrous-NO complex was rapid (<2 s) and caused a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay could occur several times with no adverse affect on its subsequent formation or on NOS catalytic activity. Neither enzyme dilution nor NO scavengers (superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl NOS or prevented the catalytic inhibition attributed to its formation. The ferrousnitrosyl complex also formed in unfractionated cell cytosol containing neuronal NOS upon initiating NO synthesis. We conclude that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrousnitrosyl complex during NO synthesis independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS.

show abstract

“…1A). Comparable Soret absorbance optima and visible band changes have been reported for low-spin six-coordinate Fe(III)⅐NO complexes of other hemeproteins (38,(55)(56)(57)(58)(59)(60). Interestingly, these results contrast with the effect of halides such as Cl Ϫ , a preferred substrate, on the Soret and visible regions of the absorbance spectrum of MPO-Fe(III), where only nominal changes were noted (53).…”

Section: Discussionmentioning

confidence: 71%

Nitric Oxide Modulates the Catalytic Activity of Myeloperoxidase

Abu-Soûd¹,

Hazen²

2000

Journal of Biological Chemistry

179

View full text Add to dashboard Cite

Myeloperoxidase (MPO), an abundant protein in neutrophils, monocytes, and subpopulations of tissue macrophages, is believed to play a critical role in host defenses and inflammatory tissue injury. To perform these functions, an array of diffusible radicals and reactive oxidant species may be formed through oxidation reactions catalyzed at the heme center of the enzyme. Myeloperoxidase and inducible nitric-oxide synthase are both stored in and secreted from the primary granules of activated leukocytes, and nitric oxide (nitrogen monoxide; NO) reacts with the iron center of hemeproteins at near diffusion-controlled rates. We now demonstrate that NO modulates the catalytic activity of MPO through distinct mechanisms. NO binds to both ferric (Fe(III), the catalytically active species) and ferrous (Fe(II)) forms of MPO, generating stable low-spin sixcoordinate complexes, MPO-Fe(III)⅐NO and MPOFe(II)⅐NO, respectively. These nitrosyl complexes were spectrally distinguishable by their Soret absorbance peak and visible spectra. Stopped-flow kinetic analyses indicated that NO binds reversibly to both Fe(III) and Fe(II) forms of MPO through simple one-step mechanisms. The association rate constant for NO binding to MPO-Fe(III) was comparable to that observed with other hemoproteins whose activities are thought to be modulated by NO in vivo. In stark contrast, the association rate constant for NO binding to the reduced form of MPO, MPO-Fe(II), was over an order of magnitude slower. Similarly, a 2-fold decrease was observed in the NO dissociation rate constant of the reduced versus native form of MPO. The lower NO association and dissociation rates observed suggest a remarkable conformational change that alters the affinity and accessibility of NO to the distal heme pocket of the enzyme following heme reduction. Incubation of NO with the active species of MPO (Fe(III) form) influenced peroxidase catalytic activity by dual mechanisms. Low levels of NO enhanced peroxidase activity through an effect on the rate-limiting step in catalysis, reduction of Compound II to the ground-state Fe(III) form. In contrast, higher levels of NO inhibited MPO catalysis through formation of the nitrosyl complex MPO-Fe(III)-NO. NO interaction with MPO may thus serve as a novel mechanism for modulating peroxidase catalytic activity, influencing the regulation of local inflammatory and infectious events in vivo.

show abstract

[15] Purification of bacterial cytochrome P-450

Cited by 76 publications

References 4 publications

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus

Neuronal Nitric Oxide Synthase Self-inactivates by Forming a Ferrous-Nitrosyl Complex during Aerobic Catalysis

Nitric Oxide Modulates the Catalytic Activity of Myeloperoxidase

Contact Info

Product

Resources

About