Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition

Abstract

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min−1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.

Assembly of The Mitochondrial Complex I Assembly Complex Suggests a Regulatory Role for Deflavination

“…The resulting cryo‐EM map at 7.8 Å resolution displayed clear secondary structural features consistent with the VLCAD‐based homology model, thus validating this procedure (Figure 3 E). Moreover, the resulting map revealed that the FAD binding site is clearly empty in the ECSIT‐bound state (Figure 3 F), in contrast to other cryo‐EM reconstructions of flavoproteins at similar resolution [36, 37] and to the crystal structure of FAD‐bound VLCAD (PDB ID:3B96 [34] ) filtered to the resolution of our cryo‐EM map (Figure S13), corroborating the role of ECSIT in promoting ACAD9 deflavination and thereby, its loss of enzymatic activity.…”

Assembly of The Mitochondrial Complex I Assembly Complex Suggests a Regulatory Role for Deflavination

“…Ther esulting cryo-EM map at 7.8 resolution displayed clear secondary structural features consistent with the VLCAD-based homology model, thus validating this procedure ( Figure 3E). Moreover,t he resulting map revealed that the FADb inding site is clearly empty in the ECSIT-bound state ( Figure 3F), in contrast to other cryo-EM reconstructions of flavoproteins at similar resolution [36,37] and to the crystal structure of FAD-bound VLCAD( PDB ID:3B96 [34] )f iltered to the resolution of our cryo-EM map ( Figure S13), corroborating the role of ECSIT in promoting ACAD9 deflavination and thereby,i ts loss of enzymatic activity.…”

An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

“…We initially took the crystal structure of Bacillus megaterium cytochrome P450 BM3 (P450 BM3 ) 30 , a non-stoichiometric complex of two CYP HEME domains and an FMN domain, as a template, for modeling the CYP 1A1–CPR complex but, despite extensive MD simulation, we did not succeed in obtaining ET-competent complexes. Indeed, considering the P450 BM3 structure as a template for modeling mammalian CYP–CPR interactions has four major drawbacks: (i) The crystal structure of P450 BM3 (PDB ID: 1BVY) is not consistent with measured ET rates due to the high redox center separation distance; (ii) The low sequence identity between P450 BM3 and CYP/CPR of about 30% for the CYP and FMN domains; (iii) Unlike mammalian CYPs, the dimeric form of P450 BM3 is catalytically functional and ET from FMN to HEME is proposed to occur in a trans fashion 31 , 32 ; (iv) Mammalian CYP and CPR are membrane-anchored proteins, whereas P450 BM3 is not and the membrane attachment may affect the binding of CPR to the CYP. Hence, it is preferable to employ a de novo protocol rather than a template-based method for modeling full-length CYP–CPR interactions in a membrane.…”