Neutron crystallography and quantum chemical analysis of bilin reductase PcyA mutants reveal substrate and catalytic residue protonation states.

PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH+) in the wildtype (WT) PcyA-BV complex, and a nearby catalytic residue Asp105 was found to have two conformations (protonated and deprotonated). Semiempirical calculations have suggested that the protonation states of BV are reflected in the absorption spectrum of the WT PcyA-BV complex. In the previously determined absorption spectra of the PcyA D105N and I86D mutants, complexed with BV, a peak at 730 nm, observed in the WT, disappeared and increased, respectively. Here, we performed neutron crystallography and quantum chemical analysis of the D105N-BV and I86D-BV complexes to determine the protonation states of BV and the surrounding residues and study the correlation between the absorption spectra and protonation states around BV. Neutron structures elucidated that BV in the D105N mutant is in a neutral state, whereas that in the I86D mutant is dominantly in a protonated state. Glu76 and His88 showed different hydrogen bonding with surrounding residues compared with WT PcyA, further explaining why D105N and I86D have much lower activities for phycocyanobilin synthesis than the WT PcyA. Our quantum mechanics/molecular mechanics calculations of the absorption spectra showed that the spectral change in D105N arises from Glu76 deprotonation, consistent with the neutron structure. Collectively, our findings reveal more mechanistic details of bilin pigment biosynthesis.

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps.

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Šresolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome.

Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ''closed-open transition'' of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conformation, makes a stable complex with heme-HO-1 and can support the HO reaction, although its efficiency is extremely limited. Furthermore, we determined the crystal structure of the CPR variant in complex with heme-HO-1 at 4.3-Å resolution. The crystal structure of a complex of CPR and its redox partner was previously unidentified. The distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme.

Distal regulation of heme binding of heme oxygenase-1 mediated by conformational fluctuations.

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes the oxidative degradation of heme. Since free heme is toxic to cells, rapid degradation of heme is important for maintaining cellular health. There have been useful mechanistic studies of the HO reaction based on crystal structures; however, how HO-1 recognizes heme is not completely understood because the crystal structure of heme-free rat HO-1 lacks electron densities for A-helix that ligates heme. In this study, we characterized conformational dynamics of HO-1 using NMR to elucidate the mechanism by which HO-1 recognizes heme. NMR relaxation experiments showed that the heme-binding site in heme-free HO-1 fluctuates in concert with a surface-exposed loop and transiently forms a partially unfolded structure. Because the fluctuating loop is located over 17 Å distal from the heme-binding site and its conformation is nearly identical among different crystal structures including catalytic intermediate states, the function of the loop has been unexamined. In the course of elucidating its function, we found interesting mutations in this loop that altered activity but caused little change to the conformation. The Phe79Ala mutation in the loop changed the conformational dynamics of the heme-binding site. Furthermore, the heme binding kinetics of the mutant was slower than that of the wild type. Hence, we concluded that the distal loop is involved in the regulation of the conformational change for heme binding through the conformational fluctuations. Similar to other enzymes, HO-1 effectively promotes its function using the identified distal sites, which might be potential targets for protein engineering.

Insights into the Proton Transfer Mechanism of a Bilin Reductase PcyA Following Neutron Crystallography.

Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis.

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin-related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 (+) sectors, ACD2 functions cell autonomously, implicating a pro-death ACD2 substrate as being cell non-autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light-dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro-death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.

Discrimination between CO and O(2) in heme oxygenase: comparison of static structures and dynamic conformation changes following CO photolysis.

Heme oxygenase (HO) catalyzes heme degradation, one of its products being carbon monoxide (CO). It is well known that CO has a higher affinity for heme iron than does molecular oxygen (O(2)); therefore, CO is potentially toxic. Because O(2) is required for the HO reaction, HO must discriminate effectively between CO and O(2) and thus escape product inhibition. Previously, we demonstrated large conformational changes in the heme-HO-1 complex upon CO binding that arise from steric hindrance between CO bound to the heme iron and Gly-139. However, we have not yet identified those changes that are specific to CO binding and do not occur upon O(2) binding. Here we determine the crystal structure of the O(2)-bound form at 1.8 Å resolution and reveal the structural changes that are specific to CO binding. Moreover, difference Fourier maps comparing the structures before and after CO photolysis at <160 K clearly show structural changes such as movement of the distal F-helix upon CO photolysis. No such changes are observed upon O(2) photolysis, consistent with the structures of the ligand-free, O(2)-bound, and CO-bound forms. Protein motions even at cryogenic temperatures imply that the CO-bound heme-HO-1 complex is severely constrained (as in ligand binding to the T-state of hemoglobin), indicating that CO binding to the heme-HO-1 complex is specifically inhibited by steric hindrance. The difference Fourier maps also suggest new routes for CO migration.

Caveolin-1 is a competitive inhibitor of heme oxygenase-1 (HO-1) with heme: identification of a minimum sequence in caveolin-1 for binding to HO-1.

Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 µM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.

Structural insights into vinyl reduction regiospecificity of phycocyanobilin:ferredoxin oxidoreductase (PcyA).

Phycocyanobilin:ferredoxin oxidoreductase (PcyA) is the best characterized member of the ferredoxin-dependent bilin reductase family. Unlike other ferredoxin-dependent bilin reductases that catalyze a two-electron reduction, PcyA sequentially reduces D-ring (exo) and A-ring (endo) vinyl groups of biliverdin IXalpha (BV) to yield phycocyanobilin, a key pigment precursor of the light-harvesting antennae complexes of red algae, cyanobacteria, and cryptophytes. To address the structural basis for the reduction regiospecificity of PcyA, we report new high resolution crystal structures of bilin substrate complexes of PcyA from Synechocystis sp. PCC6803, all of which lack exo-vinyl reduction activity. These include the BV complex of the E76Q mutant as well as substrate-bound complexes of wild-type PcyA with the reaction intermediate 18(1),18(2)-dihydrobiliverdin IXalpha (18EtBV) and with biliverdin XIIIalpha (BV13), a synthetic substrate that lacks an exo-vinyl group. Although the overall folds and the binding sites of the U-shaped substrates of all three complexes were similar with wild-type PcyA-BV, the orientation of the Glu-76 side chain, which was in close contact with the exo-vinyl group in PcyA-BV, was rotated away from the bilin D-ring. The local structures around the A-rings in the three complexes, which all retain the ability to reduce the A-ring of their bound pigments, were nearly identical with that of wild-type PcyA-BV. Consistent with the proposed proton-donating role of the carboxylic acid side chain of Glu-76 for exo-vinyl reduction, these structures reveal new insight into the reduction regiospecificity of PcyA.

Expression, purification and preliminary X-ray crystallographic analysis of cyanobacterial biliverdin reductase.

Biliverdin reductase (BVR) catalyzes the conversion of biliverdin IX α to bilirubin IX α with concomitant oxidation of an NADH or NADPH cofactor. This enzyme also binds DNA and enhances the transcription of specific genes. Recombinant cyanobacterial BVR was overexpressed in Escherichia coli, purified and crystallized. A native data set was collected to 2.34 Šresolution on beamline BL38B1 at SPring-8. An SeMet data set was collected from a microcrystal (300×10×10 µm) on the RIKEN targeted protein beamline BL32XU and diffraction spots were obtained to 3.0 Šresolution. The native BVR crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a=58.8, b=88.4, c=132.6 Å. Assuming that two molecules are present in the asymmetric unit, VM (the Matthews coefficient) was calculated to be 2.37 Å3 Da(-1) and the solvent content was estimated to be 48.1%. The structure of cyanobacterial BVR may provide insights into the mechanisms of its enzymatic and physiological functions.

Crystal structure of rat haem oxygenase-1 in complex with ferrous verdohaem: presence of a hydrogen-bond network on the distal side.

HO (haem oxygenase) catalyses the degradation of haem to biliverdin, CO and ferrous iron via three successive oxygenation reactions, i.e. haem to alpha-hydroxyhaem, alpha-hydroxyhaem to alpha-verdohaem and alpha-verdohaem to ferric biliverdin-iron chelate. In the present study, we determined the crystal structure of ferrous alpha-verdohaem-rat HO-1 complex at 2.2 A (1 A=0.1 nm) resolution. The overall structure of the verdohaem complex was similar to that of the haem complex. Water or OH- was co-ordinated to the verdohaem iron as a distal ligand. A hydrogen-bond network consisting of water molecules and several amino acid residues was observed at the distal side of verdohaem. Such a hydrogen-bond network was conserved in the structures of rat HO-1 complexes with haem and with the ferric biliverdin-iron chelate. This hydrogen-bond network may act as a proton donor to form an activated oxygen intermediate, probably a ferric hydroperoxide species, in the degradation of alpha-verdohaem to ferric biliverdin-iron chelate similar to that seen in the first oxygenation step.

Recent Advances in the Understanding of the Reaction Chemistries of the Heme Catabolizing Enzymes HO and BVR Based on High Resolution Protein Structures.

In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme Heme Oxygenase (HO) to the linear tetrapyrrole Biliverdin IXα (BV), and BV is then converted into bilirubin by Biliverdin Reductase (BVR). HO utilizes three Oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.

Conformational Equilibrium of NADPH-Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction.

Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme-HO-1, whereas the reduction in heme-HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme-HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme-HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme-HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme-HO-1 in solution. We conclude that the "open-close" transition of CPR is indispensable for electron transfer from CPR to heme-HO-1.

Functional diversification of two bilin reductases for light perception and harvesting in unique cyanobacterium Acaryochloris marina MBIC 11017.

Bilin pigments play important roles for both light perception and harvesting in cyanobacteria by binding to cyanobacteriochromes (CBCRs) and phycobilisomes (PBS), respectively. Among various cyanobacteria, Acaryochloris marina MBIC 11017 (A. marina 11017) exceptionally uses chlorophyll d as the main photosynthetic pigment absorbing longer wavelength light than the canonical pigment, chlorophyll a, indicating existence of a system to sense longer wavelength light than others. On the other hand, A. marina 11017 has the PBS apparatus to harvest short-wavelength orange light, similar to most cyanobacteria. Thus, A. marina 11017 might sense longer wavelength light and harvest shorter wavelength light by using bilin pigments. Phycocyanobilin (PCB) is the main bilin pigment of both systems. Phycocyanobilin:ferredoxin oxidoreductase (PcyA) catalyzes PCB synthesis from biliverdin via the intermediate 181 ,182 -dihydrobiliverdin (181 ,182 -DHBV), resulting in the stepwise shortening of the absorbing wavelengths. In this study, we found that A. marina 11017 exceptionally encodes two PcyA homologs, AmPcyAc and AmPcyAp. AmPcyAc is encoded on the main chromosome with most photoreceptor genes, whereas AmPcyAp is encoded on a plasmid with PBS-related genes. High accumulation of 181 ,182 -DHBV for extended periods was observed during the reaction catalyzed by AmPcyAc, whereas 181 ,182 -DHBV was transiently accumulated for a short period during the reaction catalyzed by AmPcyAp. CBCRs could sense longer wavelength far-red light through 181 ,182 -DHBV incorporation, whereas PBS could only harvest orange light through PCB incorporation, suggesting functional diversification of PcyA as AmPcyAc and AmPcyAp to provide 181 ,182 -DHBV and PCB to the light perception and harvesting systems, respectively.

Involvement of metals in enzymatic and nonenzymatic decomposition of C-terminal alpha-hydroxyglycine to amide: an implication for the catalytic role of enzyme-bound zinc in the peptidylamidoglycolate lyase reaction.

The peptide C-terminal amide group essential for the full biological activity of many peptide hormones is produced by consecutive actions of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-alpha-hydroxyglycine to an amidated peptide and glyoxylate. PAL contains 1 mol of zinc, but its role, catalytic or structural, has not yet been clarified. In this study, we found that a series of transition metals, Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), catalyze the nonenzymatic decomposition of the hydroxyglycine intermediate in a concentration-dependent manner. The second-order rate constant of the metal catalysis increased with elevation of pH, indicating that the hydrated metal acts as a general base. Extensive removal of the enzyme-bound metals remarkably diminished the PAL activity; k(cat) of the metal-depleted enzyme retaining 0.1 mol of zinc decreased to 3.2 s(-1) from 25.7 s(-1) of the wild-type enzyme. Among a series of divalent metals tested, Zn(2+), Co(2+), and Cd(2+) could fully restore the PAL activity of the metal-depleted enzyme. Especially, Zn substitution reproduced the steady-state parameters of the wild-type enzyme. On the other hand, Co and Cd substitution largely altered the kinetic parameters; the k(cat) increased 3- and 5-fold and the K(m) for the substrate increased 2.5- and 4-fold, respectively. These observations support that the enzyme-bound zinc plays a catalytic role, rather than a structural role, in the PAL reaction through the action of zinc-bound water as a general base.

Bilin-metabolizing enzymes: site-specific reductions catalyzed by two different type of enzymes.

In mammals, the green heme metabolite biliverdin is converted to a yellow anti-oxidant by NAD(P)H-dependent biliverdin reductase (BVR), whereas in O2-dependent photosynthetic organisms it is converted to photosynthetic or light-sensing pigments by ferredoxin-dependent bilin reductases (FDBRs). In NADP+-bound and biliverdin-bound BVR-A, two biliverdins are stacked at the binding cleft; one is positioned to accept hydride from NADPH, and the other appears to donate a proton to the first biliverdin through a neighboring arginine residue. During the FDBR-catalyzed reaction, electrons and protons are supplied to bilins from ferredoxin and from FDBRs and waters bound within FDBRs, respectively. Thus, the protonation sites of bilin and catalytic residues are important for the analysis of site-specific reduction. The neutron structure of FDBR sheds light on this issue.

Crystal structure of a NADPH-cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP+ binding.

Heme oxygenase-1 (HMOX1) catalyzes heme degradation utilizing reducing equivalents supplied from NADPH-cytochrome P450 reductase (CYPOR). Recently, we determined the complex structure of NADP+ -bound open-conformation stabilized CYPOR and heme-HMOX1, but the resolution was limited to 4.3 Å. Here, we determined the crystal structure of the fusion protein of open-conformation stabilized CYPOR and heme-HMOX1 at 3.25 Å resolution. Unexpectedly, no NADP+ was bound to this fusion protein in the crystal. Structural comparison of the NADP+ -bound complex and the NADP+ -free fusion protein suggests that NADP+ binding regulates the conformational change in the FAD-binding domain of CYPOR. As a result of this change, the FMN-binding domain of CYPOR approaches heme-bound HMOX1 upon NADP+ binding to enhance the electron-transfer efficiency from FMN to heme.