P450 catalysis requires two electron transfer (ET) steps. The first reduces Fe(III) to Fe(II) which enables the oxy complex, Fe(III)-OO-, to form. In the second step, reduction of the oxy-complex, ET is accompanied by proton transfer which generates the dihydroperoxy, Fe(III)-OOH2, intermediate. This is followed by heterolytic cleavage of the O-O bond to generate the active substrate oxidant, Fe(IV)=O. A central question in P450 catalysis is how protons are delivered to the oxy-complex in the second ET step. With the well studied P450cam system, crystallographic and spectroscopic studies have shown that binding of the Fe2S2 redox partner, Pdx, shifts P450cam to a more open conformation which releases the critical Asp251 to participate in the required proton delivery to the iron-linked dioxygen. Only Pdx can trigger this structural change which explains the long sought allosteric/effector role of Pdx. A closely related P450, CYP101D1, catalyzes exactly the same camphor hydroxylation reaction and has the same critical Asp residue as P450cam. However, CYP101D1 is promiscuous with respect to redox partner and, unlike P450cam, does not require its natural redox partner for catalysis. A series of mutagenesis, crystallographic, kinetic, and computational studies comparing these two P450s supports the hypothesis that Pdx binding to P450cam is required to “loosen” the structure which triggers the formation of the proton relay network while in CYP101D1 the proton relay network is preset and does not require the assistance of redox partner binding. Unexpectedly we found that converting the essential Asp to Glu eliminates activity in P450cam but not CYP101D1. In Cyp101D1 this mutation increases in the kinetic solvent isotope effect and changes the rate limiting step to reduction of the oxy-complex thereby providing a unique experimental window into proton coupled electron transfer.

Conformational dynamics and proton coupled electron transfer in P450 catalysis

ACS Fall 2021

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