Dataset Information

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

ABSTRACT: Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms.

SUBMITTER: McIntosh JA

PROVIDER: S-EPMC4635421 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

McIntosh John A JA Heel Thomas T Buller Andrew R AR Chio Linda L Arnold Frances H FH

Journal of the American Chemical Society 20150923 43

Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron ...[more]

PMID: 26299431

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Project description:It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450 → P420 transition. Here, we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm⁻¹, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm⁻¹ is also inconsistent with a generalization of the suggestion that the 221 cm⁻¹ Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm⁻¹ mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine-bound heme systems. Additionally, the inverse correlation plots of the νFe-His and νCO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO-bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of an HXC-Fe motif in many CYP systems allows the C → H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix, and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found), a somewhat larger conformational change would be necessary to recuit a nearby histidine.

Dataset Information

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

Publications

Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450.

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