Chemical mechanisms for cytochrome P-450 oxidation: spectral and catalytic properties of a manganese-substituted protein.

Abstract

Bacterial cytochrome P450 induced by camphor (PA450cam) is reconstituted with manganese-protoporphyrin IX, yielding an enzyme that displays unique spectral properties relative to previously characterized manganese-porphyrin systems. The nitric oxide complex of the manganese(II)-protein shows a hyper-metalloporphyrin spectrum suggestive of thiolate ligation to the porphyrin-bound manganese ion. In the presence of iodosobenzene as a source of active oxygen, manganese-substituted cytochrome P-450O serves-as a catalyst for the epoxidation of an enzyme-bound olefin substrate. This reactivity proceeds through a spectrally detectabk intermediate that resembles the manganese(V)-oxo complexes that have been well documented with model systems employing artificial manganese-metalloporphyrins in organic solution. Interestingly, manganese-substituted cytochrome-P-450,, shows no hydroxylation activity either in the reconstituted camphor hydroxylase system with pyridine nucleotide or in the presence of iodosobenzene and the Mn(HI) form of the protein.A wide variety of biological oxidations are catalyzed by the cytochrome P-450 monoxygenase system whereby molecular dioxygen bond scission and two reducing equivalents are coupled to substrate oxygenation and water formation (1, 2). Bacterial cytochrome PA40 induced by camphor (PA45R,), isolated from Pseudomonas putida, catalyzes both the S-exohydroxylation of camphor and the exo-epoxidation of dehydrocamphor (3) in the presence of oxygen, NADH, and two additional protein components (putidaredoxin and NADH putidaredoxin reductase) that supply the P-450 with the necessary two electrons required for the overall reaction. One feature that distinguishes cytochrome P450 from other b-type hemoproteins is the apparent presence ofan axially bound thiolate ligand to the heme iron (4).. Thiolate coordination is thought to be responsible for the unusual spectral properties of the P450 enzymes in comparison to nitrogen-coordinated hemoproteins such as hemoglobin and horseradish peroxidase (5-9). Transfer of oxygen to substrate (R) has been written as a single step but it is likely to be composed of at least two distinct reactions (11, 12). The Fe(III)Ph4PorCl/iodosobenzene system is also reported to be effective in catalyzing hydrocarbon oxygenation reactions (14,15). Recently, the higher oxidation states of iron metalloporphyrins have been partially characterized, although it remains to be established whether the oxidation equivalents in the iron-oxo species reside on the metal, the porphyrin, or both (16). These oxygenating intermediates ofthe iron systems are considerably more reactive than the chromium or manganese analogs, with chemical characterization requiring experimentation at very low temperature.Attempts to observe a higher valence state ofthe cytochrome P450 hemoprotein so far have been unsuccessful, although a variety of studies suggests intermediates on the reaction path to substrate oxygenation (17-21). A possible link between the studies with model porphyrin systems ...

NO binding to Mn-substituted homoprotocatechuate 2,3-dioxygenase: relationship to O2 reactivity

“…To our knowledge, this is the first reported occurrence of the reaction between Mn II and NO to form a stable Mn-NO protein species in a nonheme enzyme. Nitric oxide will form MnNO complexes in Mn-substituted heme proteins [22]. Also, nitric oxide will react with the Mn cluster of photosystem II and with the dinuclear Mn cluster of Mn-catalase [23], but for these enzymes the reaction occurs with Mn at an oxidation state higher than +2, and the addition of NO results in Mn reduction rather than oxidation as observed here.…”

The Second Step of the Nitric Oxide Synthase Reaction: Evidence for Ferric-Peroxo as the Active Oxidant

“…Mn-PPIX has a much lower redox potential than Fe-PPIX and has been used to directly observe high-valent states of P450s. 28 However, Mn substituted peroxidases have very little activity due to enhanced stability of the porphyrin-bound peroxide, leading to little formation of the Mn V dO intermediate during steady-state turnover. 29 Consistent with this stability, no detectable peroxidase activity is observed with Mn-iNOS heme .…”

Advances in Engineered Hemoproteins that Promote Biocatalysis

“…They are able to insert oxygen atoms into C-H bonds, which comprise some of the most difficult chemical transformations that are performed by enzymes. Interestingly, P450cam substituted with manganese protoporphyin IX does not hydroxylate its native substrate even though the spectral evidence suggests the presence of a Mn(IV)-oxo species upon reaction with iodosobenzene [75]. Comparatively to P450 enzymes, myoglobin functions as a dioxygen storage protein and does not participate in oxidative reactivity even though both proteins comprise the same heme cofactor.…”

“…Cytochromes P450 are the only heme enzymes that perform monooxygenation of unactivated alkanes [72][73][74][75][76]. They are able to insert oxygen atoms into C-H bonds, which comprise some of the most difficult chemical transformations that are performed by enzymes.…”