NADH-cytochrome B5 Oxidoreductase Research Articles

The N-terminal intrinsically disordered region of Ncb5or docks with the cytochrome b5 core to form a helical motif that is of ancient origin.

NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5 ) and cytochrome b5 reductase (b5 R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5 R domains (CS/b5 R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34 MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25-129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116 QMDWLKLTRT126 ) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes.

Ordered Motions in the Nitric-Oxide Dioxygenase Mechanism of Flavohemoglobin and Assorted Globins with Tightly Coupled Reductases.

Nitric-oxide dioxygenases (NODs) activate and combine O2 with NO to form nitrate. A variety of oxygen-binding hemoglobins with associated partner reductases or electron donors function as enzymatic NODs. Kinetic and structural investigations of the archetypal two-domain microbial flavohemoglobin-NOD have illuminated an allosteric mechanism that employs selective tunnels for O2 and NO, gates for NO and nitrate, transient O2 association with ferric heme, and an O2 and NO-triggered, ferric heme spin crossover-driven, motion-controlled, and dipole-regulated electron-transfer switch. The proposed mechanism facilitates radical-radical coupling of ferric-superoxide with NO to form nitrate while preventing suicidal ferrous-NO formation. Diverse globins display the structural and functional motifs necessary for a similar allosteric NOD mechanism. In silico docking simulations reveal monomeric erythrocyte hemoglobin alpha-chain and beta-chain intrinsically matched and tightly coupled with NADH-cytochrome b5 oxidoreductase and NADPH-cytochrome P450 oxidoreductase, respectively, forming membrane-bound flavohemoglobin-like mammalian NODs. The neuroprotective neuroglobin manifests a potential NOD role in a close-fitting ternary complex with membrane-bound NADH-cytochrome b5 oxidoreductase and cytochrome b5. Cytoglobin interfaces weakly with cytochrome b5 for O2 and NO-regulated electron-transfer and coupled NOD activity. The mechanistic model also provides insight into the evolution of O2binding cooperativity in hemoglobin and a basis for the discovery of allosteric NOD inhibitors.

Structural Characterization of Ncb5or, a Multi‐Domain Ferric Reductase Implicated in Diseases

Ncb5or (NADH cytochrome b5 oxidoreductase) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD‐Sgt1 (CS) domain, plus a 50‐residue N‐terminal region (N‐term). Ncb5or mediates electron transfer from NADH or NADPH to a redox substrate via FAD and heme cofactors in the b5R and b5 domains, respectively. The inter‐domain interactions are essential to Ncb5or redox function. Although full‐length Ncb5or from human has proven resistant to crystallization to date, we succeeded in obtaining high‐resolution structures of the b5 domain (PDB # 3LF5) and a fragment containing the CS and b5R domains in complex with NAD+ (6MV1) or NADP+ (6MV2). Despite the same general fold, the b5 domain exhibits substantially different heme ligation from that of microsomal cytochrome b5 (Cyb5A). Similarly, the b5R domain differs from microsomal cytochrome b5 reductase (Cyb5R3) in having several multi‐residue deletions and insertions that support extensive interactions with CS domain and reflect a closer relationship to Cyb5R proteins from plants, fungi and protists than to Cyb5R3 from animals. Results from electron transfer analysis using various Ncb5or fragments and mismatched Cyb5A and Cyb5R3 proteins suggest that the presence of N‐term and CS domain facilitate the specific b5‐b5R interaction in Ncb5or. In this study, we aim to characterize the structural impact of the N‐term to the b5 domain. The N‐term of Ncb5or has no homologs in animals and contains a distinctive, conserved L34MDWIRL40 motif. Circular Dichroism (CD) data revealed that the N‐term of human Ncb5or is natively disordered, but exhibits significant helical content when naturally fused with the b5 domain. Signals in the near‐UV region further indicated the presence of a tryptophan side chain in a well‐defined environment, suggesting a role for Trp37 in nucleating N‐term helical structure. This is supported by the CD data of the Trp37Ala mutant in an N‐term/b5 construct. The latter was resistant to crystallization, we therefore sought its homolog from plants, the reduced lateral root formation (RLF) protein that comprises a cytochrome b5 domain preceded by a 130‐residue N‐terminal region. There is a 52% sequence identity (74% similarity) when considering the b5 cores and the preceding 22 residues of the N‐terminal regions. A recombinant rice RLF protein comprising residues 100–218 crystallized readily, and the resultant high‐resolution structure showed Trp120 (corresponding to Trp37 in Ncb5or) to be part of an 11‐residue α‐helix (S116QMDWLKLTRT126) packing against two of the four helices that surround heme (α1 and α4). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues (Tyr85 and Tyr88 in α1, Cys124 in α4, and Leu46 in Ncb5or). The location of the N‐term helix suggests a role in mediating electron flow from NAD(P)H to its as yet unidentified redox substrate.Support or Funding InformationSupported by NIH (P30GM110761, P.I.: R.P. Hanzlik) and KU College of Liberal Arts and Sciences.

Crystal structures of the naturally fused CS and cytochrome b5 reductase (b5R) domains of Ncb5or reveal an expanded CS fold, extensive CS-b5R interactions and productive binding of the NAD(P)+ nicotinamide ring.

Ncb5or (NADH-cytochrome b5 oxidoreductase), a cytosolic ferric reductase implicated in diabetes and neurological diseases, comprises three distinct domains, cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) domain, and a novel 50-residue N-terminal region. Understanding how interdomain interactions in Ncb5or facilitate the shuttling of electrons from NAD(P)H to heme, and how the process compares with the microsomal b5 (Cyb5A) and b5R (Cyb5R3) system, is of interest. A high-resolution structure of the b5 domain (PDB entry 3lf5) has previously been reported, which exhibits substantial differences in comparison to Cyb5A. The structural characterization of a construct comprising the naturally fused CS and b5R domains with bound FAD and NAD+ (PDB entry 6mv1) or NADP+ (PDB entry 6mv2) is now reported. The structures reveal that the linker between the CS and b5R cores is more ordered than predicted, with much of it extending the β-sandwich motif of the CS domain. This limits the flexibility between the two domains, which recognize one another via a short β-sheet motif and a network of conserved side-chain hydrogen bonds, salt bridges and cation-π interactions. Notable differences in FAD-protein interactions in Ncb5or and Cyb5R3 provide insight into the selectivity for docking of their respective b5 redox partners. The structures also afford a structural explanation for the unusual ability of Ncb5or to utilize both NADH and NADPH, and represent the first examples of native, fully oxidized b5R family members in which the nicotinamide ring of NAD(P)+ resides in the active site. Finally, the structures, together with sequence alignments, show that the b5R domain is more closely related to single-domain Cyb5R proteins from plants, fungi and some protists than to Cyb5R3 from animals.

2152-P: Impaired Iron-Sulfur-Cluster Redox Regulation in Monogenic Ncb5or Diabetes

NADH cytochrome b5 oxidoreductase (Ncb5or) is a ferric reductase widely expressed in animal cells. Ncb5or deficiency results in altered iron metabolism, mitochondrial dysfunction and proliferation, and increased oxidative and endoplasmic reticulum (ER) stress. Ncb5or null (KO) mice present early-onset lean diabetes, ataxia and hearing loss. Similar clinical manifestations are observed in patients with type 2 Wolfram Syndrome that is caused by mutations in CISD2 (CDGSH iron-sulfur domains 2). Its [2Fe-2S] iron-sulfur-cluster (ISC) is redox sensitive and becomes labile when oxidized. Because both proteins are associated with ER, Ncb5or may function as a CISD2 reductase for ISC redox regulation. This hypothesis appears to be supported by our recent data. When fed a chow admixture with 400 ppm pioglitazone, a FDA-approved drug capable of stabilizing FeII-ISC in vitro, Ncb5or KO mice exhibit a 4-week delay in developing overt diabetes, specifically, at age 11 weeks vs. 7 weeks under chow. Pioglitazone results in a significant increase in the content of FeII-ISC CISD2 among the total in the liver of prediabetic KO mice when compared to wild type (WT) counterparts, namely, a KO/WT ratio of 0.6 post pioglitazone vs. 0.3 under chow (by native gel electrophoresis and immuno-blot analyses with antibodies that preferentially recognize CISD2 containing FeII-ISC). Pioglitazone also reduces oxidative and ER stress response in the liver of KO mice (qRT-PCR of MT’s, ATF3, and XBP1-spliced transcripts) and attenuates mitochondrial dysfunction in KO mouse embryonic fibroblasts (extracellular flux analysis). However, iron dyshomeostasis, as indicated by transcript profiles of iron genes in KO cells and splenomegaly in KO mice, is not rescued by pioglitazone. Our findings suggest that Ncb5or maintains the redox status of ISC in CISD2 and that, through stabilizing ferrous ISC, pioglitazone can partially rescue defects in Ncb5or-null cells. Studies are underway to characterize the Ncb5or-CISD2 axis in beta-cells. Disclosure H. Zhu: None. A. Muto: None. T. Home: None. W. Wang: None. F. Urano: Board Member; Self; Healthbeat. Research Support; Self; Aetas, Amylyx, JDRF, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health. Stock/Shareholder; Self; CytRx. Other Relationship; Self; Novus Biologicals. Funding University of Kansas Medical Center Research Institute

NCB5OR Deficiency in the Cerebellum and Midbrain Leads to Dehydration and Alterations in Thirst Response, Fasted Feeding Behavior, and Voluntary Exercise in Mice

Cytosolic NADH-cytochrome-b5-oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues. We have previously reported that global ablation of NCB5OR in mice results in early-onset lean diabetes with decreased serum leptin levels and increased metabolic and feeding activities. The conditional deletion of NCB5OR in the mouse cerebellum and midbrain (conditional knock out, CKO mice) results in local iron dyshomeostasis and altered locomotor activity. It has been established that lesion to or removal of the cerebellum leads to changes in nutrient organization, visceral response, feeding behavior, and body weight. This study assessed whether loss of NCB5OR in the cerebellum and midbrain altered feeding or metabolic activity and had an effect on serum T3, cortisol, prolactin, and leptin levels. Metabolic cage data revealed that 16 week old male CKO mice had elevated respiratory quotients and decreased respiratory water expulsion, decreased voluntary exercise, and altered feeding and drinking behavior compared to wild-type littermate controls. Most notably, male CKO mice displayed higher consumption of food during refeeding after a 48-h fast. Echo MRI revealed normal body composition but decreased total water content and hydration ratios in CKO mice. Increased serum osmolality measurements confirmed the dehydration status of male CKO mice. Serum leptin levels were significantly elevated in male CKO mice while prolactin, T3, and cortisol levels remain unchanged relative to wild-type controls, consistent with elevated transcript levels for leptin receptors (short form) in the male CKO mouse cerebellum. Taken together, these findings suggest altered feeding response post starvation as a result of NCB5OR deficiency in the cerebellum.

Loss of NCB5OR in the cerebellum disturbs iron pathways, potentiates behavioral abnormalities, and exacerbates harmaline-induced tremor in mice.

Iron dyshomeostasis has been implicated in many diseases, including a number of neurological conditions. Cytosolic NADH cytochrome b5 oxidoreductase (NCB5OR) is ubiquitously expressed in animal tissues and is capable of reducing ferric iron in vitro. We previously reported that global gene ablation of NCB5OR resulted in early-onset diabetes and altered iron homeostasis in mice. To further investigate the specific effects of NCB5OR deficiency on neural tissue without contributions from known phenotypes, we generated a conditional knockout (CKO) mouse that lacks NCB5OR only in the cerebellum and midbrain. Assessment of molecular markers in the cerebellum of CKO mice revealed changes in pathways associated with cellular and mitochondrial iron homeostasis. (59)Fe pulse-feeding experiments revealed cerebellum-specific increased or decreased uptake of iron by 7 and 16weeks of age, respectively. Additionally, we characterized behavioral changes associated with loss of NCB5OR in the cerebellum and midbrain in the context of dietary iron deprivation-evoked generalized iron deficiency. Locomotor activity was reduced and complex motor task execution was altered in CKO mice treated with an iron deficient diet. A sucrose preference test revealed that the reward response was intact in CKO mice, but that iron deficient diet consumption altered sucrose preference in all mice. Detailed gait analysis revealed locomotor changes in CKO mice associated with dysfunctional proprioception and locomotor activation independent of dietary iron deficiency. Finally, we demonstrate that loss of NCB5OR in the cerebellum and midbrain exacerbated harmaline-induced tremor activity. Our findings suggest an essential role for NCB5OR in maintaining both iron homeostasis and the proper functioning of various locomotor pathways in the mouse cerebellum and midbrain.

Cytosolic localization of NADH cytochrome b₅ oxidoreductase (Ncb5or).

Acyl-CoA desaturation in the endoplasmic reticulum (ER) membrane depends on cytosolic NADH or NADPH, whereas NADPH in the ER lumen is utilized by prereceptor glucocorticoid production. It was assumed that NADH cytochrome b5 oxidoreductase (Ncb5or) might connect Acyl-CoA desaturation to ER luminal redox. We aimed to clarify the ambiguous compartmentalization of Ncb5or and test the possible effect of stearoyl-CoA on microsomal NADPH level. Amino acid sequence analysis, fluorescence microscopy of GFP-tagged protein, immunocytochemistry, and western blot analysis of subcellular fractions unequivocally demonstrated that Ncb5or, either endogenous or exogenous, is localized in the cytoplasm and not in the ER lumen in cultured cells and liver tissue. Moreover, the involvement of ER-luminal reducing equivalents in stearoyl-CoA desaturation was excluded.

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase

NADH cytochrome b5 oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.

High fat feeding exacerbates endoplasmic reticulum stress and beta cell demise

A growing body of evidence suggests that the insulin-producing beta cells in the pancreas are susceptible to saturated fatty acid-induced endoplasmic reticulum (ER) stress and apoptosis, and that this may contribute to the development of diabetes. In this issue of the European Journal of Lipid Science and Technology, Guo et al. [p. 233–234] report on accelerated beta cell dysfunction and death induced by high fat feeding of the Ncb5or knockout mouse, which lacks the ER-associated NADH-cytochrome b5 oxidoreductase and has impaired fatty acid desaturation.

Beta‐cell injury in Ncb5or‐null mice is exacerbated by consumption of a high‐fat diet

NADH-cytochrome b5 oxidoreductase (Ncb5or) in endoplasmic reticulum (ER) is involved in fatty acid metabolism, and Ncb5or(-/-) mice fed standard chow (SC) are insulin-sensitive but weigh less than wild type (WT) littermates. Ncb5or(-/-) mice develop hyperglycemia at about age 7 weeks due to β-cell dysfunction and loss associated with saturated fatty acid accumulation and manifestations of ER and oxidative stress. Here we report that when Ncb5or(-/-) mice born to heterozygous mothers fed a high fat (HF) diet continue to ingest HF, they weigh as much as SC-fed WT at age 5 weeks. By age 7 weeks, diabetes mellitus develops in all HF-fed vs. 68% of SC-fed Ncb5or(-/-) mice. Islet β-cell content in age 5-week Ncb5or(-/-) mice fed HF for 7 days is lower (53%) than for those fed SC (63%), and both are lower than for WT (75%, SC, vs. 69%, HF). Islet transcript levels for markers of mitochondrial biogenesis (PGC-1α) and ER stress (ATF6α) are higher in Ncb5or(-/-) than WT mice but not significantly affected by diet. Consuming a HF diet exacerbates Ncb5or(-/-) β-cell accumulation of intracellular saturated fatty acids and increases the frequency of ER distention from 11% (SC) to 47% (HF), thus accelerates β-cell injury in Ncb5or(-/-) mice.

Development of diabetes in lean Ncb5or-null mice is associated with manifestations of endoplasmic reticulum and oxidative stress in beta cells

NADH-cytochrome b5 oxidoreductase (Ncb5or) is an endoplasmic reticulum (ER)-associated redox enzyme involved in fatty acid metabolism, and phenotypic abnormalities of Ncb5or −/− mice include diabetes and lipoatrophy. These mice are lean and insulin-sensitive but become hyperglycemic at age 7 weeks as a result of β-cell dysfunction and loss. Here we examine early cellular and molecular events associated with manifestations of β-cell defects in Ncb5or −/− mice. We observe lower islet β-cell content in pancreata at age 4 weeks and prominent ER distention in β-cells by age 5 weeks. Ultrastructural changes progress rapidly in severity from age 5 to 6 weeks, and their frequency rises from 10% of β-cells at 5 weeks to 33% at 6 weeks. These changes correlate temporally with the onset of diabetes. ER stress responses and lipid load in Ncb5or −/− β-cells were assessed with isolated islets from mice at age 5 weeks. Expression levels of the stress marker protein Grp78/BiP and of phosphorylated eIF2α protein were found to be reduced, although their transcript levels did not decline. This pattern stands in contrast to the canonical unfolded protein response. Ncb5or −/− β-cells also accumulated higher intracellular levels of palmitate and other free fatty acids and exhibited greater reactive oxygen species production than wild-type cells. An alloxan-susceptible genetic background was found to confer accelerated onset of diabetes in Ncb5or −/− mice. These findings provide the first direct evidence that manifestations of diabetes in lean Ncb5or −/− mice involve saturated free fatty acid overload of β-cells and ER and oxidative stress responses.

Structural basis of inter‐domain electron transfer in human Ncb5or

NADH cytochrome b5 oxidoreductase (Ncb5or) is found in all animals and contains three domains: cytochrome b5 (b5), CHORD‐SGT1 (CS) and b5 reductase (b5R). The two redox domains are homologous to microsomal b5 (Cyb5A) and b5R (Cyb5R3), the two known electron donors of stearoyl‐CoA desaturase in vitro. To elucidate the structural basis of inter‐domain electron transfer in human Ncb5or, we generated individual b5 and b5R domains for structural and kinetic studies. The crystal structure of Ncb5or‐b5 was solved by X‐ray diffraction with a resolution of 1.95 Å. There are fewer surface charged residues in Ncb5or‐b5 than Cyb5A, suggesting a weaker electrostatic interaction in b5‐b5R than Cyb5A‐Cyb5R3. This was confirmed in our measurements of electron transfer in b5‐b5R, Cyb5‐Cyb5R3 and mismatched pairs. The linkage through CS likely obviates the need for strong interactions between b5 and b5R in Ncb5or. Ncb5or‐b5 has two helix‐loop‐helix motifs surrounding heme, but differs from Cyb5A in that helix 4 is canted at 45º and shorter. The heme‐ligating His112 residue between helix 3 and 4 is displaced due to one‐residue deletion (before) and insertion (after). Trp114 in helix 4 of Ncb5or‐b5 is a well conserved surface residue potentially involved in inter‐domain recognition or electron transfer. We thus conclude that the Ncb5or‐b5 core has a conserved b5 scaffold with unique features. (Supported by NIH)

Characterization of inter‐domain electron transfer in Ncb5or, a redox enzyme involved in fatty acid desaturation

Microsomal cytochrome b5 (b5) and cytochrome b5 reductase (b5R) are redox proteins that donate electrons to stearoyl‐CoA desaturase (SCD) in fatty acid desaturation. NADH cytochrome b5 oxidoreductase (Ncb5or) contains b5 and b5R domains, and a linkage domain similar to the CS (CHORD‐SGT1) family members. Our recent studies show that Ncb5or functions as an alternative electron donor for SCD. Ncb5or knockout mice display lipoatrophy and diabetes as a result of impaired fatty acid desaturation. In this study, we aim to characterize the properties of individual human Ncb5or domains, the inter‐domain electron transfer and the role of CS domain. Recombinant b5, b5R, and CS+b5R domains were generated from a bacterial system. Native gel electrophoresis revealed that b5 is stable under heat treatment, but b5R and CS+b5R are not. Inter‐domain electron transfer can be reconstituted by mixing b5 with b5R or CS+b5R under excess NADH, indicating a non‐essential role of CS in electron transfer. The percentage of heme reduction increases with decreasing salt concentrations, suggesting an electrostatic interaction. In the same buffer, b5R reduces more heme than CS+b5R. Both b5R and CS+b5R alone can reduce cytochrome c with the same rate. The addition of b5 increases the rate, more with b5R than CS+b5R. We conclude that electrons can flow from both b5 and b5R to cytochrome c but only the b5‐b5R interaction is specific. Supported by NIH.

Proton Translocation Linked to the Activity of the Bi-Trans-membrane Electron Transport Chain

Recently we have proposed and presented evidence suggesting the existence of a "bi-trans-membrane" electron transport chain, located at the contact sites between outer and inner mitochondrial membranes, which can be utilized to promote either the oxidation of exogenous NADH in the presence of catalytic amounts of added cytochrome c or the reduction of exogenous cytochrome c supported by the oxidation of respiratory substrates present inside the mitochondria. Here we show that the oxidation of exogenous NADH is accompanied by a net alkalinization of the incubation medium preceded by a transient acidification phase. In oxygen-pulse experiments, the alcohol oxidation (induced by the addition of alcohol dehydrogenase) was used to mimic a cytosolic source of reducing equivalents. Oxygen pulses promote an acidification-alkalinization proton cycle which is insensitive to antimycin and myxothiazol inhibitory effect, is stimulated by valinomycin, inhibited by trypsin-aprotinin complex, abolished by the protonophore carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and is absent or at least inverted (alkalinization-acidification cycle) in broken mitochondria. The oxidation of cytosolic substrates, mediated by the bi-trans-membrane electron transport chain, does not involve endogenous cytochrome c and is associated with a vectorial proton translocation from the inside to the outside of the mitochondria. In the out → in electron transport pathway the components involved appear to be cytosolic reduced substrates → NADH produced by cytosolic dehydrogenases activity → NADH-cytochrome b5 oxidoreductase complex leaning out the external side of the external membrane → exogenous cytochrome c → cytochrome oxidase of contact sites → molecular oxygen. The possible components of the in → out pathway are matrix respiratory substrates → primary dehydrogenases of the matrix → Complexes I, II, and III of the respiratory chain present in the inner membrane → NADH-cytochrome b5 oxidoreductase system of the external membrane → exogenous cytochrome c → additional cytosolic electron accepters or, alternatively, cytochrome oxidase of contact sites. The two pathways can be considered a bi-trans-membrane electron channeling system which, at the level of bridges set up by the contact points between the outer and the inner mitochondrial membrane, may represent a Link between the redox processes occurring inside with those present outside the mitochondrion.

Myoglobin: cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase.

Metmyoglobin (metMb) reduction by metMb reductase from heart muscle requires cytochrome b5 as electron-transfer mediator. The existence of a metMb-ferrous cytochrome b5 complex is demonstrated by mutual perturbation of the proteins' respective electrophoretic titration curves between pH 4 and 7. The same technique shows a preferential binding of cytochrome b5 over metMb by the enzyme. The paramagnetic hyperfine shifts in the cytochrome b5 1H NMR spectrum are perturbed by metMb, indicating the formation of a specific bimolecular complex with a 1:1 stoichiometry and a binding constant estimated to be less than 10 microM. The resonances assigned to the cytochrome b5 heme 6-propionate methylene group exhibit the largest complexation shifts. Computer modeling implicates lysines 47, 50, and 98 of metMb as contact points with cytochrome b5 carboxylate residues 43, 44, 60, and heme 6-propionate. The mechanism of the enzymatic reduction establishes metMb reductase as an NADH-cytochrome b5 oxidoreductase. Cytochrome b5 is reduced at near diffusion-controlled rates by the enzyme with a turnover number of 1000 min-1 X Km for the cytochrome is 0.9 microM versus 100 microM reported for the erythrocyte enzyme. Ferrous cytochrome b5 then reduces metMb nonenzymatically with an apparent rate constant of 4.9 X 10(4) M-1 min-1 X Acetylation of metMb, which does not affect its oxygen affinity or chemical reduction, renders it a poor substrate for enzymatic reduction. This study suggests a function for the three exterior lysine residues conserved in all mammalian myoglobin sequences: they are contact points for complexation with cytochrome b5.

In vitro synthesis and post-translational insertion into microsomes of the integral membrane protein, NADH-cytochrome b5 oxidoreductase.

RNA extracted from a free polysome fraction from rat liver was used to direct translation in nuclease-treated rabbit reticulocyte lysates, and the [35S]methionine-labelled, in vitro-synthesized, cytochrome b5 reductase was isolated with specific antibodies. Analysis by SDS-polyacrylamide gel electrophoresis, non-equilibrium pH gradient electrophoresis and one-dimensional peptide mapping failed to reveal any difference between the in vitro-synthesized reductase and the enzyme endogenous to rat liver microsomes. To study the integration of the in vitro-synthesized reductase into membranes, carboxypeptidase Y was used as a proteolytic probe. The reductase endogenous to rat liver microsomes was resistant to attack by carboxypeptidase Y, but was degraded to a smaller form when the microsomes were solubilized by detergent. Likewise, the enzyme synthesized in vitro was attacked by carboxypeptidase Y, but became largely resistant after post-translational incubation with dog pancreatic microsomes, indicating that an integration into membranes similar to the physiological one had occurred. It is concluded that cytochrome b5 reductase is probably not synthesized as a precursor and inserts post-translationally into the membrane. The results are discussed in relation to the particular subcellular distribution of the reductase and to the possible topology in the lipid bilayer of its C-terminal non-polar membrane-binding segment.

Isolation of NADH-cytochrome B5 oxidoreductase from rat liver microsomes

1. NADH-cytochrome b 5 oxidoreductase is released from rat liver microsomes when the latter are incubated at 37° with a lysosomal fraction from rat liver. 2. Release is maximal when equal amounts of microsomes and the lysosomal fraction a on protein basis are incubated. The release is optimal at pH 5.5 and is unaffected by iodoacetamide. 3. A 3-4-fold activation of NADH-cytochrome b 5 oxidoreductase activity occurs during release from microsomes but NADH-ferricyanide oxidoreductase activity, although released, is not activated. 4. A 20-fold purified protease fraction from lysosomes, containing cathepsin D activity, is 20 times more active in releasing the oxidoreductase than is the original lysosomal fraction. 5. A simple procedure, involving ultrafiltration, molecular sieving and isoelectric focussing, is described for isolating the oxidoreductase as a single protein species.