Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.

Bonfils, Claude; Balny, Claude; Maurel, Patrick

doi:10.1016/s0021-9258(19)68784-3

Cited by 106 publications

(27 citation statements)

References 39 publications

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“…A role for b 5 was proposed to be an alternate electron donor for reduction of the P450 ferrous-dioxygen complex, thus promoting active oxygen (ferryl-oxo P450) formation (20)(21)(22)(23)(24). Reduction of the P450 LM2 ferrous-dioxygen complex by b 5 was confirmed by rapid mixing experiments (25,26). Loss of b 5 effects upon reconstitution with redox-inactive heme analogues also supported this finding (16,(27)(28)(29).…”

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confidence: 75%

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Perret

Pompon

1998

Biochemistry

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Contradictory mechanisms involving conformational or redox effects have been proposed for the enhancement of cytochrome P450 activities by cytochrome b5 in reconstituted systems. These mechanisms were reinvestigated for human liver P450 3A4 bound to recombinant yeast membranes including human P450 reductase and various levels of human b5. Species conversions were calculated on the basis of substrate, oxygen, and electronic balances in six different substrate conditions. Electron flow from P450 reductase to ferric 3A4 was highly dependent on the nature of substrate but not on the presence of b5. P450 uncoupling by hydrogen peroxide formation was decreased by b5, leading to a corresponding increase in the rate of ferryl-oxo complex formation. Nevertheless, the major b5 effects mainly relied on an increased partition of ferryl-oxo complex to substrate oxidation compared to reduction to water, which could support a conformation change based mechanism. However, further steady-state investigations evidenced that electron carrier properties of b5 were strictly required for this modulation and that redox state of b5 was ruled by the nature and concentration of 3A4 substrates. Moreover, rapid kinetic analysis of b5 reduction following NADPH addition suggested that b5 was reduced by the 3A4 ferrous-dioxygen complex and reoxidized by subsequent P450 oxygenated intermediates. A kinetic model involving a 3A4-b5 electron shuttle within a single productive P450 cycle was designed and adjusted. This model semiquantitatively simulated all presented experimental data and can be made compatible with the effect of the redox-inactive b5 analogue previously reported in reconstituted systems. In this model, synchronization of the b5 and 3A4 redox cycles, binding site overlap between b5 and reductase, and dynamics of the b5-3A4 complex were critical features. This model opened the way for designing complementary experiments for unification of b5 action mechanisms on P450s.

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confidence: 75%

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Perret

Pompon

1998

Biochemistry

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“…39 In summary, earlier functional studies showed the principal role of Cytb 5 on P450 catalysis by CYP17A1 is as an electron donor. 24,40,41 The rR studies reported herein provide structural evidence for an additional allosteric role when 17OH progesterone (OH-PROG) is the substrate for carbon−carbon bond scission. This additional allosteric enhancement is not expected, and not seen, when 17OH-PREG is at the active site.…”

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confidence: 79%

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b₅

Liu

Denisov²,

Sligar³

et al. 2021

J. Am. Chem. Soc.

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CYP17A1 is an essential human steroidogenic enzyme, which catalyzes two sequential reactions leading to the formation of androstenedione from progesterone and dehydroepiandrosterone from pregnenolone. The second reaction is the C17− C20 bond scission, which is strongly dependent on the presence of cytochrome b 5 and displays a heretofore unexplained more pronounced acceleration when 17OH-progesteone (17OH-PROG) is a substrate. The origin of the stimulating effect of cytochrome b 5 on C−C bond scission catalyzed by CYP17A1 is still debated as mostly due to either the acceleration of the electron transfer to the P450 oxy complex or allosteric effects of cytochrome b 5 favoring active site conformations that promote lyase activity. Using resonance Raman spectroscopy, we compared the effect of Mn-substituted cytochrome b 5 (Mn-Cytb 5 ) on the oxy complex of CYP17A1 with both proteins co-incorporated in lipid nanodiscs. For CYP17A1 with 17OH-PROG, a characteristic shift of the Fe− O mode is observed in the presence of Mn-b 5 , indicating reorientation of a hydrogen bond between the 17OH group of the substrate from the terminal to the proximal oxygen atom of the Fe−O−O moiety, a configuration favorable for the lyase catalysis. For 17OHpregnenolone, no such shift is observed, the favorable H-bonding orientation being present even without Mn-Cytb 5 . These new data provide a precise allosteric interpretation for the more pronounced acceleration seen for the 17OH-PROG substrate.

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“…Alternatively, we can also use a rate constant of 3 s –1 for cytochrome b 5 reduction of the cytochrome P450 2B4 ferric-superoxo intermediate. In this case, Trp hydroxylation would still moderately compete with its nitration by a 1:12 ratio. − Therefore, the ability of TxtE to resist the reduction of the ferric-superoxo intermediate appears to improve the efficiency of nitration activity by avoiding access to intermediates that lead to substrate hydroxylation.…”

Section: Discussionmentioning

confidence: 99%

The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

et al. 2021

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TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO2-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO2-Trp under single- or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (FeII) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet–visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity.

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Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.

Cited by 106 publications

References 39 publications

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b₅

The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

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Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.

Cited by 106 publications

References 39 publications

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb5Rules Uncoupling Mechanisms

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb5Rules Uncoupling Mechanisms

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b5

The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide

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Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Electron Shuttle between Membrane-Bound Cytochrome P450 3A4 andb₅Rules Uncoupling Mechanisms

Substrate-Specific Allosteric Effects on the Enhancement of CYP17A1 Lyase Efficiency by Cytochrome b₅