Djemel Hamdane

Abstract Proton conductive materials are of significant importance and highly desired for clean energy-related applications. Discovery of practical metal-organic frameworks (MOFs) with high proton conduction remains a challenge due to the use of toxic chemicals, inconvenient ligand preparation and complication of production at scale for the state-of-the-art candidates. Herein, we report a zirconium-MOF, MIP-202(Zr), constructed from natural α-amino acid showing a high and steady proton conductivity of 0.011 S cm −1 at 363 K and under 95% relative humidity. This MOF features a cost-effective, green and scalable preparation with a very high space-time yield above 7000 kg m −3 day −1 . It exhibits a good chemical stability under various conditions, including solutions of wide pH range and boiling water. Finally, a comprehensive molecular simulation was carried out to shed light on the proton conduction mechanism. All together these features make MIP-202(Zr) one of the most promising candidates to approach the commercial benchmark Nafion.

Neuroglobin and cytoglobin reversibly bind oxygen in competition with the distal histidine, and the observed oxygen affinity therefore depends on the properties of both ligands. In the absence of an external ligand, the iron atom of these globins is hexacoordinated. There are three cysteine residues in human neuroglobin; those at positions CD7 and D5 are sufficiently close to form an internal disulfide bond. Both cysteine residues in cytoglobin, although localized in other positions than in human neuroglobin, may form a disulfide bond as well. The existence and position of these disulfide bonds was demonstrated by mass spectrometry and thiol accessibility studies. Mutation of the cysteines involved, or the use of reducing agents to break the S-S bond, led to a decrease in the observed oxygen affinity of human neuroglobin by an order of magnitude. The critical parameter is the histidine dissociation rate, which changes by about a factor of 10. The same effect is observed with human cytoglobin, although to a much lesser extent (less than a factor of 2). These results suggest a novel mechanism for the regulation of oxygen binding; contact with an appropriate electron donor would provoke the release of oxygen. Hence the oxygen affinity would be directly linked to the redox state of the cell.

NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR) 4The abbreviations used are: CYPOR, NADPH-cytochrome P450 oxidoreductase; cyt, cytochrome; FADhq, FAD hydroquinone; FADsq, FAD semiquinone; FADox, oxidized FAD; FMNhq, FMN hydroquinone; FMNsq, FMN semiquinone; FMNox, oxidized FMN; NOS, nitric oxide synthase; r.m.s.d., root mean square deviation; ΔTG, the 2 (ΔThr-236, Gly-237) amino acid deletion mutant; ΔTGEE, the 4 (ΔThr-236, Gly-237, Gly-238, Gly-239) amino acid deletion mutant. is a ∼78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH → FAD → FMN to members of the ubiquitous cytochrome P450 superfamily (1.Masters B.S. Okita R.T. Pharmacol. Ther. 1980; 9: 227-244Crossref PubMed Scopus (43) Google Scholar, 2.Paine M.J. Scrutton N.S. Munro A.W. Gutierrez A. Robert G.C.K. Wolf C.R. Ortiz de Montellano P.R. Cytochrome P450. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 115-148Crossref Scopus (91) Google Scholar). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3.Schacter B.A. Nelson E.B. Marver H.S. Masters B.S. J. Biol. Chem. 1972; 247: 3601-3607Abstract Full Text PDF PubMed Google Scholar), cytochrome b5 (4.Enoch H.G. Strittmatter P. J. Biol. Chem. 1979; 254: 8976-8981Abstract Full Text PDF PubMed Google Scholar), and cytochrome c (5.Horecker B.L. J. Biol. Chem. 1950; 183: 593-605Abstract Full Text PDF Google Scholar). The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe+3OO)2- cyt P450 intermediate (6.Ortiz de Montellano P.R. De Voss J.J. Ortiz de Montellano P.R. Cytochrome P450. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 183-245Crossref Scopus (207) Google Scholar). In vivo, CYPOR cycles between the one- and three-electron reduced forms (7.Iyanagi H.S. PubMed Scopus Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the reduced form is an it is the FMN the that donates an electron to its redox partners M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). CYPOR is the of the enzyme PubMed Scopus Google Scholar), A. A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and a in the of M.J. J. Wolf C.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). CYPOR is also a it Pharmacol. PubMed Scopus Google Scholar). CYPOR of an its to the and the of cytochrome structures of the form of the wild-type and are B.S. A. Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The amino of the domain are to and FMN the of the protein of a and domain with sequence and structural to oxidoreductase A connecting a sequence and the FMN and FAD and is the of the FMN and FAD In the a FMN. In the wild-type the two are in electron transfer B.S. A. Scopus Google Scholar). on the of the two an electron transfer rate of is PubMed Scopus Google Scholar). the electron transfer rate between the two is J.J. PubMed Scopus Google Scholar, A. Wolf C.R. Scrutton N.S. PubMed Scopus Google Scholar). rate and of electron transfer in a that electron transfer is likely the in the are in is to with mutagenesis that the amino acid on the FMN are involved in with cyt P450 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The structural cyt P450 interact with the FMN domain of CYPOR provided by the of a complex between the heme and of cyt P450 A. PubMed Scopus Google Scholar). In the of FMN are the heme on the of cyt P450 three the electron the mutagenesis and the of the complex between the heme and FMN of cyt P450 that CYPOR a rearrangement in the course of electrons from NADPH to cyt P450. In structures of CYPOR that the FMN domain is with to the rest of the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the a to interact with its redox partners to the of a structural in the that the and the of the on the hinge between the FMN and the connecting it is and in the The and sequence of the hinge been by and the of the on the of been The demonstrate that lengthening the linker its sequence the of CYPOR. In deletion of amino electron transfer from FAD to FMN, the ability of the FMN domain to electrons to cyt P450 The hinge deletion variant has been crystallized in three of with cyt P450. of CYPOR Cytochrome c and ability of CYPOR hinge to cytochrome c and in at as J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the are provided in the that the of cytochrome c activity with the two and amino acid deletion to a of from the CYPOR, the of cytochrome c also in the of FMN and The the activity of the CYPOR. The of FMN and FAD J. Biol. Chem. Full Text PDF PubMed Google Scholar). The and cytochrome c and NADPH by the of cytochrome c at of cytochrome c and and NADPH and The by the rate of the the of by CYPOR by the of the at CYPOR with oxidized at The reaction by the of NADPH to a of The of the reduced an at of J. Biol. Chem. Full Text PDF PubMed Google Scholar). of by Cyt P450 P450 and as PubMed Scopus Google in The of at by as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the of CYPOR by at a with a in an as M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). are in the of the of of Ferric Cyt P450 by CYPOR in the and of rate of of cyt P450 in the of by wild-type, ΔTG, and CYPOR as M.J. J. Biol. Chem. 1979; 254: Full Text PDF PubMed Google Scholar). are in the of the of by Cyt P450 in the of and rate of of and by cyt P450 in the of the reduced CYPOR at and a as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a protein complex between oxidized and cyt P450 in the of at a of The to the protein at a of the cyt complex reduced with to the ferrous cyt P450 and to the reduced CYPOR. The the cyt complex of the and with of from The reaction with at different The and the in as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by a a and form of the to as the in the of the the of ΔTGEE, and in a similar to that of the protein in The in also similar that and an at the to and of FMN and to the protein by two cycles of to The protein to in the by of the protein and 2 of and a in a and then in to A at the with the PubMed Scopus Google Scholar). The to the with of a and c and and three of the the in protein Biol. PubMed Scopus Google J. PubMed Scopus Google Scholar), when the wild-type CYPOR used as the when of CYPOR, the FAD domain and the FMN used as three FAD and two FMN with the of and with an of all the from the the electron FAD and FMN at the of the The FMN domain at a an FMN domain in the FAD and one to the then the linker between the two of the second in the In the of in all three FAD the PubMed Scopus Google and model with P. Biol. PubMed Scopus Google Scholar), of and and the the three FAD and two FMN used the of of and and all three FAD are one FMN domain is and the of the and of the second FMN domain all to from the of in the from the hinge that to the and of the FMN and the FMN domain FMN domain in the The and and the are in and in are the c of in an of a of in the FAD in are the of a of in the in a of CYPOR and Cyt P450 A. Scopus Google and J. Biol. Scholar). In the A as the and cyt P450 as the of A and of the and of cyt P450 to in the of CYPOR as involved in the with cyt P450 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of cyt P450 been by mutagenesis to involved in the with CYPOR J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of A and and of cyt P450 as in the the from the in of the in the the FMN to the of the the hinge the FMN to the connecting domain of CYPOR in the FMN and FAD of the protein electron transfer from FAD FMN to the protein cyt the hinge the of the hinge a sequence of the known CYPOR and a structural of two CYPOR structures and B.S. A. Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google The the of the linker and that it from to in the the sequence between and is the structures Full Text Full Text PDF PubMed Scopus Google Scholar). has an acid hinge, the electron the to an J. A. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The hinge sequence is with to the hinge of CYPOR. the of the CYPOR hinge electron the the ability to its electron transfer partner, cyt the of the hinge by two and and by two and amino protein The hinge also two and form with the of in the FMN the of on the of the and to In a with amino acid in the of the hinge → → → and → to amino and the sequence of the with Cyt P450 Cytochrome and ability of the CYPOR to by the physiological redox partner, cyt P450 by the of to The and and and the and The acid deletion to as a wild-type rate of the activity of the 4-amino acid deletion to as of the wild-type and CYPOR with redox partners The activity of with redox partners cytochrome and cyt P450 at as The activity of CYPOR with cyt P450 is in of of of cyt in a Cytochrome c is a physiological redox CYPOR. Nevertheless, it is used to the activity of the and of the J. Biol. Chem. Full Text PDF PubMed Google Scholar). is that the cyt P450 and cytochrome on the FMN domain of the is Pharmacol. Google Scholar, PubMed Scopus Google Scholar). The in 2 demonstrate that the and wild-type activity with cytochrome that the two the activity of the In the and a in activity with the wild-type that the of cytochrome c by the of the hinge in the wild-type CYPOR. In the ΔTG, and ΔTGEE, amino acid deletion reduced cytochrome c and The activity of the deletion with cytochrome c to a of the cytochrome c The of cytochrome c the wild-type and and deletion and 2 NADPH to wild-type, and the and 4-amino acid deletion with a of and the a cytochrome c and lengthening nor the sequence of the hinge with the of the the hinge by two amino to its with the cyt P450 of it its activity with cytochrome c from a of to The 4-amino acid deletion in the hinge a to the activity with cyt P450 and cytochrome The activity with cyt P450 and cytochrome c activity of the wild-type The domain in a protein, NOS, has a hinge between its FMN and connecting domain when by two amino to nitric oxide to a rate of electron transfer to the heme J. A. PubMed Scopus Google Scholar). a of the hinge in CYPOR and is to the to transfer electrons to physiological redox partners. with CYPOR is with the that in redox proteins the activity of the by allowing Full Text Full Text PDF PubMed Scopus Google Scholar). of Cyt P450 in the activity of with cyt P450 and cytochrome the activity in to the of deletion mutant. the of in the three different and in an the of A and the FAD of and C and of of the FMN domain of and most of the of the C FMN domain the FMN of and C FMN are it is that the and the of in of the and two are the same as of the in the wild-type the of the the FMN domain of C and its FMN of electron are the wild-type CYPOR in the hinge is in all three are of the electron to the are in In the FAD of the wild-type and three extended structures are that the and of the FMN are The that the of of the three FAD of the CYPOR are the same as that of wild-type is with the that the activity of is the same as that in The of A is the most of the three it is open the wild-type The between the and in A is that of the wild-type is The of the FMN domain with to the FAD domain in the two are The FMN domain in A has from the FAD domain and that the is to and to interact with its electron transfer partner, cyt P450. The movement of the FMN occurs by pivoting on the of of of at the of the of a FMN domain movement between the conformation of the and PubMed Google and J. PubMed Scopus Google and the other and C extended between and and with In the the FMN domain of A has with the FAD domain of and are the electron the A FMN domain with that of the protein by of the protein the of C is to the of the FMN domain as a of the the FMN domain of C in the that the is in In the wild-type FAD and FMN are and the of are in that the electron transfer between the is B.S. A. Scopus Google Scholar). the between the two of the two in A is in is and that in C is that electron transfer between the two in of three is in The activity cytochrome c in is of that in A similar in a the FMN and FAD and that of the FMN and FAD of CYPOR of the wild-type activity in cytochrome c Wolf C.R. A. PubMed Scopus Google Scholar). the activity in to a the electron transfer from FAD to FMN in a same the other it is also that, in of in electrons from FAD to FMN in the same two in the and in a rate of cytochrome c activity is that forms a in as in the of CYPOR, in of the hinge A. Gutierrez A. PubMed Scopus Google Scholar). deletion of CYPOR to form a in and its electron transfer to from the FAD domain of one to the FMN domain of the other in the the of of the and of the protein in the protein to that the protein is the wild-type CYPOR, that the a conformation with is also with the of the the wild-type protein is to the is to that the protein has a open again with the from NADPH to FAD at the in the and are three the of the to the oxygenase activity of the cyt P450 and to cytochrome is that hydride transfer from NADPH to FAD has been in of the of the CYPOR, by that the of by the similar to that of the wild-type protein The of has been used to the of hydride transfer from NADPH to it has been that FAD is the of in the protein M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). are also with the of other that a acid deletion in the hinge of CYPOR the of and NADPH A. Gutierrez A. PubMed Scopus Google Scholar). The second and most likely the activity of the in of its open is that electron transfer from FAD to FMN has been The to the of ΔTGEE, is that the FMN domain in a conformation that electrons from FAD is of to the from FAD to FMN in in FAD to FMN FMN to cyt P450 electron transfer in the the of electron transfer from the FAD to FMN by by a of In the wild-type protein, is a of electrons between the two J.J. PubMed Scopus Google Scholar, A. Wolf C.R. Scrutton N.S. PubMed Scopus Google Scholar). the of the electrons is by the redox of the and (7.Iyanagi H.S. PubMed Scopus Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of CYPOR by of and that, when at the of CYPOR by of NADPH is The of FAD by In the wild-type protein, of in also the electron transfer from to FMN. from the the rate the is similar all three that of FAD by NADPH has been in the The ability of the FAD of the proteins to at the wild-type rate the that of FAD by NADPH in a The of the second of the wild-type protein is in the the of the second is of the same as the the rate of the second are similar to the rate at the second to transfer of an electron from to of wild-type and CYPOR by and of NADPH The in at as The of the CYPOR NADPH in A and in A is the of the a in a in a in a in in a of the as by the at in the wild-type protein and with the and 4-amino acid deletion and a in wild-type, with the the is at M.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the second at in the electron transfer from in the to FMN to form an of two forms of the and of CYPOR by of of the electron transfer of the by the of of the by a of of the of CYPOR by the in the is to the the form of the to the to the that NADPH is an obligate hydride the form of the is to a second hydride from The in and that the of by a of NADPH are the by a one of Nevertheless, also demonstrate that electron transfer is in and in In the wild-type protein, electron transfer from FAD to FMN, occurs with a rate of its at The then as the is reduced by a second of with a in at the of The rate of by the second of NADPH is with the two The is of a The FAD transfer its electrons to a FMN, has been reduced by the The second is the in FAD with the oxidized by is and its a at the of is with that of with of to by the The of the to the is three of in the wild-type The of the deletion intermediate between of and The of the of of the at by a of NADPH to electron transfer as the reduced and The of of NADPH the of the of of wild-type CYPOR at a its rate the same as that at one of NADPH and similar to the of the to at of at the to of the form of the by a second of of in and of transfer from FAD to FMN and in the 2 and occurs at a rate the rate of cytochrome c and 2 and The reaction at a of the rate of cytochrome c at a of protein the rate of electron transfer from FAD to FMN, an electron transfer is Ferric Cyt P450 in the of the FMN domain of in a conformation that is of electrons to cyt the of of cyt P450 with by of ferrous cyt P450. with cyt at a rate of to and to the J. Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of of cyt P450 by wild-type and in the and of The of cyt P450 by the deletion that of wild-type and with a rate similar to the rate of electron transfer from FAD to FMN. Cyt P450 in the of the the redox of the the of the reaction PubMed Scopus (43) Google Scholar). also that the the rate and the of the of the wild-type In a reduced cyt with a of the of the time FMN The of cyt P450 by of the of that the with the of the heme the of FMN. The of of cyt P450 by two rate and to the rate electron transfer to FMN in the of NADPH of and cyt P450 by CYPOR in the of a of NADPH The in at The and cyt P450 and the A is the of the in a by Cyt P450 at the as the P450 two electrons to the of The electron the cyt P450. The ferrous cyt P450 and then accepts a second electron from the FMN to form the reduced oxyferrous cyt P450 In the of cyt P450 oxyferrous cyt P450 the of to the second electron to oxyferrous cyt P450 and its ability to the two and The of is to the electron transfer and on the to transfer of the second electron to oxyferrous cyt P450 and its to cyt P450 and the The rate of in a by a complex of the reduced cyt reduced with PubMed Scopus (43) Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The reaction and the by The of and by cyt P450 in the of wild-type and are and in a It is that at and at of cyt P450 is by cyt P450 a intermediate in the of CYPOR J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus (43) Google of by cyt P450 in the of wild-type and in of of of cyt P450. The as in of of of cyt P450. The as in a the of the the of the CYPOR, an of the of and electron transfer in wild-type and and 2 2 in and in and in J.J. PubMed Scopus Google Scholar, A. Wolf C.R. Scrutton N.S. PubMed Scopus Google Scholar). 4 4 in and provided with sufficient electrons. In the of of cyt P450 to electron transfer from FAD to FMN to the rate of the same in the wild-type and The rate is the rate wild-type CYPOR PubMed Scopus (43) Google Scholar). demonstrate that the 4-amino acid deletion in the hinge the of the FMN domain with cyt provided that the FMN in the of the the extended to cyt a model complex between electron transfer the structures of A and cyt P450 PubMed Scopus Google Scholar). A is the extended of the three The from the two and is in The CYPOR has an open conformation that the of the cyt P450 the FMN of the has been by mutagenesis to involved in cyt P450 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The between the two is of is between the FMN domain and cyt P450. The between the FMN domain and cyt P450 are and are and from the FMN domain and and of cyt and one at the of cyt P450 and of The FMN domain with the of the cyt P450 that the of the heme and FMN are to other The between the heme and the of the and the of is in between the of and of cyt that as an electron transfer between the FMN and heme the of the FMN domain and cyt P450 and the of FMN and the heme in model are similar to in the of the complex between the and of cyt P450 that the between the two in the cyt P450 is A. PubMed Scopus Google Scholar). the FAD domain also with cyt P450 in the model A is the open conformation of CYPOR that the cyt P450 a electron transfer between the two partners. cyt P450 by mutagenesis to with CYPOR are on the of model between cyt P450 and the FMN domain J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the and structural presented demonstrate the time that the two in CYPOR are to by pivoting on the C terminus of the that a similar of occurs in the wild-type protein and that movement the FMN domain to a conformation of with its physiological electron transfer partner, cyt P450. The wild-type CYPOR in the two are an conformation the FAD to FMN electron the open electron transfer from the CYPOR FMN to the cyt P450 and structures the structural of the of CYPOR and demonstrate that CYPOR undergoes its J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the CYPOR is the of In the of NADPH to to and the of on the of from the FAD J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transfer from FAD to FMN is by the hinge the FMN and connecting by and the two in a CYPOR conformation electron same hinge of the FAD and FMN it also the FMN domain to on the C terminus of the hinge and a structural rearrangement that separates the FMN and FAD to form an extended conformation of with and cyt P450. The hinge of CYPOR, electron from FAD to FMN and from FMN to cyt P450 by the and of the two The three CYPOR structures the by CYPOR undergoes a in the course of electron transfer from FAD to FMN and from FMN to cyt its physiological redox CYPOR is the of the diflavin enzyme family that to the of electron transfer in other members of the and the at the in with of the of are The of in of the is with

The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp(147) and Arg(514) in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP(+) revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP(+) shows movement of the Gly(631)-Asn(635) loop. In the NADP(+)-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP(+) shows movement of the Gly(631)-Asn(635) loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly(631)-Asn(635) loop movement controls NADPH binding and NADP(+) release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.