Conformational Motion of Ferredoxin Enables Efficient Electron Transfer to Heme in the Full-Length P450_TT

Abstract

Cytochrome P450 monooxygenases (P450s) are versatile biocatalysts used in natural products biosynthesis, xenobiotic metabolisms, and biotechnologies. In P450s, the electrons required for O 2 activation are supplied by NAD(P)H through stepwise electron transfers (ETs) mediated by redox partners. While much is known about the machinery of the catalytic cycle of P450s, the mechanisms of long-range ET are largely unknown. Very recently, the first crystal structure of full-length P450 TT was solved. This enables us to decipher the interdomain ET mechanism between the [2Fe−2S]-containing ferredoxin and the heme, by use of molecular dynamics simulations. In contrast to the "distal" conformation characterized in the crystal structure where the [2Fe−2S] cluster is ∼28 Å away from heme-Fe, our simulations demonstrated a "proximal" conformation of [2Fe−2S] that is ∼17 Å [and 13.7 Å edge-to-edge] away from heme-Fe, which may enable the interdomain ET. Key residues involved in ET pathways and interdomain complexation were identified, some of which have already been verified by recent mutation studies. The conformational transit of ferredoxin between "distal" and "proximal" was found to be controlled mostly by the long-range electrostatic interactions between the ferredoxin domain and the other two domains. Furthermore, our simulations show that the full-length P450 TT utilizes a flexible ET pathway that resembles either P450 Scc or P450 cam . Thus, this study provides a uniform picture of the ET process between reductase domains and heme domain.

Structural insights into 3Fe-4S ferredoxins diversity inM.tuberculosishighlighted by a first redox complex with P450

“…The ferredoxin domain must have sufficient mobility upon reduction for the next step where it interacts with the CYP domain. The molecular dynamic simulation studies confirm the transit of ferredoxin from "distal" to "proximal" conformation enabling efficient electron transfer from the reduced ferredoxin to heme domain 38 . However, there is still the possibility of a dimeric complex (as for P450BM3 39 ) with the inter-monomer electron transfer.…”

Control of carbon monoxide dehydrogenase orientation by site-specific immobilization enables direct electrical contact between enzyme cofactor and solid surface

“…In a DET-based system, it is known that the enzyme bioactivity 16 , 17 , the surface and solution chemistry, the substrate diffusion 12 , and the ET distance at the enzyme–electrode interface 11 – 14 are among the factors that can affect the ET rate. In this study, CODH-L was designed and engineered to fuse the gbp at the N- or C- terminus, or at both termini, thereby generating various immobilization sites on the CODH-L. More specifically, the orientation of the enzyme molecules can be controlled depending on the immobilization site.…”

“…In general, redox enzymes possess average hydrodynamic diameters ranging from 55 to 150 Å (40–850 kDa), with one or more redox centers 23 that are electrically inaccessible because their active sites are buried deep beneath the surface of the protecting protein shell 24 . In a natural system, the physiological distances between the edges of the redox centers are within ~14 Å, which permits efficient electron tunneling 12 . However, electrons must often be transferred over distances >14 Å, which is typically accomplished by multistep tunneling through chains of redox centers individually positioned within the 14 Å boundary 13 .…”

Critical Roles of Exchange and Superexchange Interactions in Dictating Electron Transfer and Reactivity in Metalloenzymes

“…Likewise, the spin-regulated inner-sphere ET can be enhanced by both exchange and superexchange interactions in [Fe4S4]-dependent SAM enzymes, which enable the efficient cleavage of the SC­(γ) or SC5′ bond of SAM. In addition to inner-sphere ET, superexchange interactions may modulate the long-range ET between metalloenzymes. , As the shifting electron from the electron donor can have spin–spin interactions with the unpaired electrons on bridge or electron acceptor, the spin state of the bridge or electron acceptor may exert key effects on the kinetics of ET. In the case when the shifting electron from the electron donor is antiferromagnetically coupled to electrons of a bridge or an electron acceptor, the superexchange interactions between the donor and the acceptor/bridge may enhance the electronic coupling and thus significantly speed up electron transfer, which has been deduced from spin-regulated long-range electron transfer from CDH to LPMO.…”