Putidaredoxin

Tyson CA, ID Lipscomb, 1C Gunsalus (1972) The roles of putidaredoxin and P450, j in methylene hydroxylation. J Biol Chem 247 5777-5781. 

Cytochrome P-450 has been characterized in four stable states [Fe, Fe " RH, Fe RH, (O2—Fe ) RH (metastable)] during its oxygenase reaction cycle. In the complete native system a flavoprotein and a redoxin (putidaredoxin) act as electron donors but also as effectors that complement the cytochrome. In the more complex microsomal system the sequence and intermediates are less well defined the electron-transfer chain contains two flavoproteins and one cytochrome, whose interactions with cytochrome P-450 are still the subject of great controversy. 

Fig. 7. The effect of various substances on the spin-state equilibriutn of bacterial cytochrome P-450. The fraction of protein in the high-spin state is plotted (in arbitrary units) against the concentration (in fiM) of the natural effector putidaredoxin (Pd°) or the concentration in percent (v/v) of the organic solvents ethylene glycol or n-butanol. It is apparent that butanol induces a shift in spin state which is similar to that induced by the protein effector.

This enzyme [EC 1.14.15.1], also known as camphor 5-exo-methylene hydroxylase, and cytochrome P450-cam, catalyzes the reaction of (+)-camphor with putidare-doxin and dioxygen to generate (-F)-exo-5-hydroxy-camphor, oxidized putidaredoxin, and water. A heme-thiolate acts as a cofactor. The enzyme can also utilize ( )-camphor as a substrate, and l,2A-campholide will result in the formation of 5-exo-hydroxy-l,2-campholide. V. Ullrich W. Duppel (1975) The Enzymes, 3rd ed., 12, 253. 

In a biotransformation system designed to mediate the o-dealkylation of 7-ethoxycumarin to 7-hydroxycoumarin, the genes for the P450 from Streptomyces peucelius and putidaredoxin reductase (CamA) and putidaredoxin (CamB) from... 

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

Figure 4. Role of putidaredoxin in the methylene hydroxylation system for camphor (39)...

Adsorption of putidaredoxin on gold electrodes has been studied using dynamic spectroscopic ellipsometry and differential capacitance measurements [307]. In Ref. 307, a method for the measurement of metal surface optical perturbation during protein adsorption at a constant potential has been described. The method is based on the concept that the charged transition layer develops between the electrode substrate and the adsorbate. 

Apart from the above techniques, the electromodulated reflectance spectroscopy combined with cyclic voltammetry has been utilized by Gaigalas et al. [14] in the investigations of electron transfer between the 2Fe-2S protein putidaredoxin and either bare or bekanamycin-modified Ag electrode. Of the two models considered, the free diffusion model, as compared to the adsorbed layer model, exhibited better concordance with the experimental data. After modification of the Ag electrode with bekanamycin, it exhibited only a small increase in the observed redox reaction... 

NADH NADH-putidaredoxin reductase (fp), putidaredoxin (Fe-S) P-450 Pseudomonas putida D-camphor hydroxylase 40-42)... 

Figure 16-17 Electron paramagnetic resonance spectrum of the Fe-S protein putidaredoxin in the natural form (32S) and with labile sulfur replaced by selenium isotopes. Well-developed shoulders are seen in the low-field end of the spectrum of the 77Se (spin = l/2)-containing protein. From Orme-Johnson et al.29S Courtesy of W. H. Orme-Johnson.

One oxygen of 02 is incorporated into the hydroxyl group of the product, whereas the other is incorporated into water. The enzymes usually include a cytochrome P450, an iron-sulfur cluster-containing protein, such as adrenodoxin or putidaredoxin, and a flavoprotein reductase. 

Fig. 7. Mossbauer spectra of oxidized plant-type iron-sulfur proteins in zero applied magnetic field. Abbreviations AZI = A zotobacter Fe-S protein I, 4.6°K AZII = Azoiobacter Fe-S protein II, 4.2 °K Put. = Putidaredoxin, 4.2 °K Ad.— Pig Ad-renodoxin, 4.2 °K Clos. = Clostridial paramagnetic protein, 4.2 °K PPNR = Spinach ferredoxin, 4.5 °K Parsley = Parsley Ferredoxin, 4.2 °K. Velocity scale is relative to iron in platinum...

These spectra, taken at variable temperatures and a small polarizing applied magnetic field, show a temperature-dependent transition for spinach ferredoxin. As the temperature is lowered, the effects of an internal magnetic field on the Mossbauer spectra become more distinct until they result at around 30 °K, in a spectrum which is characteristic of the low temperature data of the plant-type ferredoxins (Fig. 11). We attribute this transition in the spectra to spin-lattice relaxation effects. This conclusion is preferred over a spin-spin mechanism as the transition was identical for both the lyophilized and 10 mM aqueous solution samples. Thus, the variable temperature data for reduced spinach ferredoxin indicate that the electron-spin relaxation time is around 10-7 seconds at 50 °K. The temperature at which this transition in the Mossbauer spectra is half-complete is estimated to be the following spinach ferredoxin, 50 K parsley ferredoxin, 60 °K adrenodoxin, putidaredoxin, Clostridium. and Axotobacter iron-sulfur proteins, 100 °K. 

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