Steady-State Kinetic Investigation of Cytochrome P450cam:  Interaction with Redox Partners and Reaction with Molecular Oxygen

Purdy, Matthew M.; Koo, Laura S.; Montellano, Paul R. Ortiz de; Klinman, Judith P.

doi:10.1021/bi0356045

Cited by 42 publications

(62 citation statements)

References 36 publications

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“…Previous work showed that the initial rate of WT P450cam varied with changing PdX and P450cam concentrations when NADH was the cofactor. [13,14] However, when either 1 a or 1 b was used, the rate of camphor oxidation was not influenced by the concentration of either PdX (range: 0.5-7.5 mm) or P450cam (range: 0.5-1.5 mm). In contrast, the initial rate increased 1.3-fold when the concentration of PdR was increased from 0.75 to 2.8 mm; this suggests that the reduction of the FAD ligand within PdR was rate-limiting when 1 a or 1 b was used, and that the rate could be further improved by using a mutant of PdR that is more active towards the NADH biomimics.…”

Section: P450mentioning

confidence: 95%

Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

2008

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Section: P450mentioning

confidence: 95%

Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

2008

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“…Pdx receives reducing equivalents from Pdr in two one-electron steps and delivers them one at a time to P450 cam . Acting as a shuttle, Pdx forms transient electron transfer (ET) complexes with its redox partners during turnover (2)(3)(4)(5)(6). The x-ray structures of all components of the camphor monooxygenase have been determined (7)(8)(9)(10), but neither Pdr⅐Pdx nor Pdx⅐P450 cam native complexes have been crystallized thus far.…”

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confidence: 99%

Crystal Structure of the Putidaredoxin Reductase·Putidaredoxin Electron Transfer Complex

Sevrioukova

Poulos

Churbanova

2010

Journal of Biological Chemistry

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In the camphor monooxygenase system from Pseudomonas putida, the [2Fe-2S]-containing putidaredoxin (Pdx) shuttles electrons between the NADH-dependent putidaredoxin reductase (Pdr) and cytochrome P450 cam . The mechanism of the Pdr⅐Pdx redox couple has been investigated by a variety of techniques. One of the exceptions is x-ray crystallography as the native partners associate weakly and resist co-crystallization. Here, we present the 2.6-Å x-ray structure of a catalytically active complex between Pdr and Pdx C73S/C85S chemically cross-linked via the Lys 409Pdr -Glu 72Pdx pair. In Pseudomonas putida, the camphor monooxygenase system uses NADH as a source of electrons and consists of three soluble proteins: FAD-containing putidaredoxin reductase (Pdr, 3 45.6 kDa), [2Fe-2S]-containing putidaredoxin (Pdx, 11.4 kDa), and cytochrome P450 cam (P450 cam , 46.6 kDa) (1). Pdx receives reducing equivalents from Pdr in two one-electron steps and delivers them one at a time to P450 cam . Acting as a shuttle, Pdx forms transient electron transfer (ET) complexes with its redox partners during turnover (2-6). The x-ray structures of all components of the camphor monooxygenase have been determined (7-10), but neither Pdr⅐Pdx nor Pdx⅐P450 cam native complexes have been crystallized thus far.The mechanism of interaction and interprotein ET in the Pdr⅐Pdx pair has been the focus of several research groups including ours. Significant progress has been made in the general understanding of how the Pdr⅐Pdx complex is formed and functions. In particular, both two-and one-electron reduced species of Pdr were identified as catalytically competent redox intermediates (3, 4, 11); ionic and hydrophobic interactions as well as steric complementarity were proven to contribute to molecular recognition between the partners (4, 12, 13) and involve Tyr 33 , Asp 38 , Arg 66 , Glu 72 , and Cys 73 of Pdx (14 -16); and, based on the x-ray structures of the flavo-and iron-sulfur proteins (8, 10, 17), a computer model for the Pdr⅐Pdx ET complex was generated and experimentally tested (16). However, the precise manner of the Pdr-Pdx interaction, the docking sites, and interface residues remained unknown. The most direct way to obtain this information would be determination of the x-ray structure of the Pdr⅐Pdx complex, but, despite our multiple attempts, the native proteins resisted co-crystallization. This could in part be due to weak association (K d ϭ 66 M) (16) and quick inactivation of wild type Pdx in solution (8).To overcome this problem, we attempted to produce a functionally active Pdr⅐Pdx conjugate. Having screened various cross-linking agents, reaction conditions, and Pdx mutants, we prepared a catalytically competent complex between wild type Pdr and Pdx C73S/C85S covalently linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) via the Lys 409Pdr -Glu 72Pdx salt bridge (6). The double mutant of Pdx was chosen for a functional analysis because substitution of cysteines 73 and 85 with serine, a highly isosteric analogue, only mildly aff...

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“…In this regard, Pochapsky et al (15) have proposed that Pdx helps prevent uncoupling of electron transfer from product formation. It has been shown that in catalysis, putidaredoxin reductase does not form a ternary complex (putidaredoxin reductase/Pdx/P450-CAM) (16). Therefore, single turnover experiments to probe the oxygenation mechanism using Pdx and P450-CAM in the absence of putidaredoxin reductase are likely to be relevant to the natural catalytic events.…”

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Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

Glascock

Ballou

Dawson

2005

Journal of Biological Chemistry

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The single turnover of (1R)(؉)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3°C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, ϳ140 and ϳ85 s ؊1, were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM⅐Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substratefree P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.The cytochrome P-450 family of heme-containing mono-oxygenases is involved in the metabolism of xenobiotics and in the production of physiologically important molecules (1). A defining characteristic of the P-450 family is the proximal thiolate-ligated heme. P450-CAM (P450-CAM, CYP101) 3 from Pseudomonas putida catalyzes the hydroxylation of (1R)(ϩ)-camphor to form (1R)(ϩ)-5-exo-hydroxycamphor (Reaction 1). The electrons required for the reaction flow from NADH to the flavoprotein, putidaredoxin reductase, then to the iron-sulfur (2Fe/2S) protein, putidaredoxin (Pdx), and finally to P450-CAM. In the widely quoted P450 reaction cycle shown in Fig. 1 (1), substrate binding converts the ferric low spin resting state (1) to the ferric high spin form (2). Electron transfer from reduced Pdx gives high spin deoxyferrous P-450 (3). Dioxygen binding yields the oxy-ferrous adduct, a ferrous-O 2 /ferric superoxide resonance hybrid (4a 7 4b). CO binding to 3 generates the ferrous-CO derivative (5). Addition of the second electron from reduced Pdx has been proposed to yield a ferric peroxo species (6a), protonation of which gives the hydroperoxo state (6b). Protonation of the distal oxygen produces water and compound I, a ferryl porphyrin -cation radical (7). This highly oxidizing intermediate abstracts a hydrogen...

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Steady-State Kinetic Investigation of Cytochrome P450cam: Interaction with Redox Partners and Reaction with Molecular Oxygen

Cited by 42 publications

References 36 publications

Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

Crystal Structure of the Putidaredoxin Reductase·Putidaredoxin Electron Transfer Complex

Direct Observation of a Novel Perturbed Oxyferrous Catalytic Intermediate during Reduced Putidaredoxin-initiated Turnover of Cytochrome P-450-CAM

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