Chemical characterization of cytochrome P-450cam

K, Dus; Katagiri, Masayuki; Yu, Chang An; Dl, Erbes; Ic, Gunsalus

doi:10.1016/0006-291x(70)90026-4

Cited by 85 publications

(16 citation statements)

References 15 publications

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“…It should also be noted that putidaredoxin, which serves as electron donor and effector in the camphor hydroxylation cycle [3], changes the high-spin/lowspin equilibrium [8] and AH (P. Debey unpublished results), probably by its polyanionic character [14].…”

Section: Discussionmentioning

confidence: 99%

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

Lange

Bonfils

Debey

1977

European Journal of Biochemistry

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The spin state of camphor-bound cytochrome P-450 is shown to depend largely on medium and temperature in aqueous as well as in mixed organic buffer. At sub-zero temperatures a variation of pa,, ionic strength or camphor concentration modifies the spin equilibrium from nearly pure high-spin form to nearly pure low-spin form. Since the apparent pK, of transition is a linear function of log I, the spin state seems to be controlled by the electrostatic potential in the heme proximity. K' is found to have a specific effect on the spin state.The change of enthalpy, AH, of the spin transition depends on the same parameters as the equilibrium constant, in the organic cosolvent as well as in aqueous buffer. As the cosolvent effect is reflected by higher AH values, and KCl and pH tend to lower AH, the cosolvent effect can easily be compensated. Therefore kinetic studies of the spin conversion might well be undertaken at sub-zero temperature in this solvent.Recently [l], we have shown the advantages of studies of the first step of the camphor hydroxylation cycle : the binding of camphor to cytochrome P-450 at sub-zero temperatures in a mixed organic solvent. Simultaneously with results of Sligar [2] above 0 "C in aqueous phosphate buffer, we found that camphorbound cytochrome P-450 (Fe3+.RH) is in thermal spin equilibrium in fluid hydro-organic medium at temperatures well below 0 "C. Some interesting results have been gathered by such a technique, for example the mechanism of camphor binding and the low-spin+high-spin transition of Fe3+ . RH were found to depend on pa, in the physiological pa, range, and the ionization state of a basic residue (possibly histidyl) was shown to be affected by the absence or presence of camphor.However, these results were overshadowed by the fact that the cosolvent, although not denaturing, shifted the [high-spin]/[low-spin] equilibrium constant K and AH to higher values [l].Here we show that K and AH of the low-spin to high-spin equilibrium of Fe3 +. RH are in fact relative values which depend on (a) physiological parameters such as ionic strength, concentration of K + , which is found to be a specific modulator of the high-spin/ low-spin equilibrium, concentration of camphor, pa, and (b) non-physiological parameters such as polyelectrolytes, organic cosolvent.The possibility of modulating K (= [high-spin]/ [low-spin]) at sub-zero temperatures by variation in these factors, namely pa, and ionic strength, enables us to study the transition of nearly pure low-spin to nearly pure high-spin Fe3+. RH and sheds some light on the structural basis of the mechanism of the spin conversion.Furthermore, we have examined the cosolvent effect on AH, a very important term of the mechanism of a reaction. In this paper we show how K may be adjusted to any desired value by simple modification of the parameters (a) or (b) and how the cosolvent effect on AH can be compensated.These various studies are the basis of a kinetic analysis of the Fe3+. RH spin state transition, which will be developed in a future pape...

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Section: Discussionmentioning

confidence: 99%

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

Lange

Bonfils

Debey

1977

European Journal of Biochemistry

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“…Putida cytochrome P450car has a molecular weight of 45,000 and consists of one polypeptide chain and one protoheme IX group (6). Its biological role is the selective hydroxylation of the 5-methylene carbon of D(+)-camphor to form the exo-5-alcohol.…”

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confidence: 99%

“…3) is typical of a low-spin ferric heme compound. Much effort has been invested in identification of the axial ligands of the heme iron in P450 (6,7). The tendency of d6-systems to take on the diamagnetic low-spin form, even in rather weak ligand fields, has long been recognized (16).…”

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confidence: 99%

Proton Magnetic Resonance Reveals High-Spin Iron (II) in Ferrous Cytochrome P450 _cam from Pseudomonas putida

Keller

Wüthrich

Debrunner

1972

Proc. Natl. Acad. Sci. U.S.A.

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“…CYP6A1 [9]. All P450s form similar secondary and tertiary structures [10][11][12] which combine with a heme cofactor that forms the catalytic reaction center. Heme comprises a central iron atom bound to a protoporphyrin ring by four nitrogen ligands forming a planar structure.…”

Section: Introductionmentioning

confidence: 99%

Strategies for the construction of insect P450 fusion enzymes

Talmann

Wiesner

Vilcinskas

2017

Zeitschrift Für Naturforschung C

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Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state. Both challenges can be addressed by creating fusion proteins that combine the P450 and CPR functions while eliminating membrane anchors, allowing the production and purification of soluble multifunctional polypeptides suitable for industrial applications. Here we discuss several strategies for the construction of fusion enzymes combining insect P450 with CPRs.

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Chemical characterization of cytochrome P-450cam

Cited by 85 publications

References 15 publications

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

Proton Magnetic Resonance Reveals High-Spin Iron (II) in Ferrous Cytochrome P450 _cam from Pseudomonas putida

Strategies for the construction of insect P450 fusion enzymes

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Chemical characterization of cytochrome P-450cam

Cited by 85 publications

References 15 publications

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450

Proton Magnetic Resonance Reveals High-Spin Iron (II) in Ferrous Cytochrome P450 cam from Pseudomonas putida

Strategies for the construction of insect P450 fusion enzymes

Contact Info

Product

Resources

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Proton Magnetic Resonance Reveals High-Spin Iron (II) in Ferrous Cytochrome P450 _cam from Pseudomonas putida