Characterization of the covalent cross-links of the active sites of amidinated cytochrome b5 and NADH:cytochrome b5 reductase

Preparations of amidinated cytochrome b5 and cytochrome b5 reductase, cross-linked by using a soluble carbodiimide to promote the formation of covalent bonds between carboxyl groups of the hemeprotein and nucleophilic residues of the flavoprotein at the surfaces involved in protein-protein contacts during electron transfer, have been used to characterize the charge pair interactions that occur during electron transfer between the free proteins. Sequence analyses of tryptic, V8 protease-, and Asp-N protease-generated peptides show that the heme propionyl carboxyl group at the surface of the cytochrome forms an ester bond with Ser162 of the reductase, thus implicating Lys163 as the normal participant in ionic bonding between the active sites of the two proteins. Moreover, Lys41 and Lys125 directly form amide bonds with carboxyl residues on the active-site surface of the cytochrome. In the case of Lys41, this involves Glu52 and/or Glu60, and Glu47 and/or Glu48 for Lys125, again implicating these residues as the groups that form charge pairs during normal interactions between the active sites of the two proteins.     

Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.     

Structure and properties of the recombinant NADH-cytochrome b5 reductase of Physarum polycephalum

A cDNA for NADH-cytochrome b(5) reductase of Physarum polycephalum was cloned from a cDNA library, and the nucleotide sequence of the cDNA was determined (accession no. AB259870). The DNA of 943 base pairs contains 5'- and 3'-noncoding sequences, including a polyadenylation sequence, and a coding sequence of 843 base pairs. The amino acid sequence (281 residues) deduced from the nucleotide sequence was 25 residues shorter than those of vertebrate enzymes. Nevertheless, the recombinant Physarum enzyme showed enzyme activity comparable to that of the human enzyme. The recombinant Physarum enzyme showed a pH optimum of around 6.0, and apparent K(m) values of 2 microM and 14 microM for NADH and cytochrome b(5) respectively. The purified recombinant enzyme showed a typical FAD-derived absorption peak of cytochrome b(5) reductase at around 460 nm, with a shoulder at 480 nm. These results suggest that the Physarum enzyme plays an important role in the organism.     

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.     

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.     

NADH binding to cytochrome b5 reductase blocks the acetylation of lysine 110

Lysine residues outside of the NADH-binding site in the soluble catalytic fragment of cytochrome b5 reductase were modified with ethyl acetimidate and acetic anhydride while the binding site was protected by formation of the stable oxidized nucleotide-reduced flavoprotein complex. This treatment had a minimal effect on enzyme activity; the turnover number with potassium ferricyanide was 45,300 in the native reductase and 39,200 in the derivative. Subsequent reaction with [3H]acetic anhydride after the removal of NADH resulted in the loss of 91% of the enzyme activity and the incorporation of 1.9 eq of acetyl groups into the protein. Treatment with 1 M hydroxylamine at pH 13 indicated that only lysine residues were acetylated, and fragmentation of the derivative with cyanogen bromide and subfragmentation with trypsin and chymotrypsin demonstrated that only Lys110 was labeled at high specific activity, with a stoichiometry of 0.83 acetyl groups/mol, in good agreement with the loss of enzyme activity observed. The remaining label was distributed at low levels among four or more additional lysine residues. These results demonstrate that only Lys110 is specifically protected by NADH and is therefore the residue which provides the epsilon-amino group implicated in NADH binding in cytochrome b5 reductase.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase

Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

Differences in C-terminal amino acid sequences between erythrocyte and liver cytochrome b5 isolated from pig and human. Evidence for two tissue-specific forms of cytochrome b5

Two forms of cytochrome b5, a soluble erythrocyte form and a membrane-bound liver form, were purified from pig and human, and structural differences between them were analyzed. Porcine and human erythrocyte cytochrome b5 consisted of 97 amino acid residues and contained the same catalytic domain structure (residues 1-96) as that of the corresponding liver cytochrome b5, but had one amino acid replacement at the C-terminus (residue 97). These results suggest that erythrocyte cytochrome b5 is not derived from the liver protein by proteolysis but a translational product from another distinct mRNA of cytochrome b5.     

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus.

Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

The reactivities of tyrosine and tryptophan residues in lipid-bound cytochrome b5.

Purified cytochrome b5 from rabbit liver microsomes was bound to liposomes prepared from microsomal lipids. Tyrosyl and tryptophyl side chains of the protein were modified by water-soluble reagents and the reactivities of these amino acid residues in the liposome-bound cytochrome b5 were compared to those of the free protein. At pH 13, 80% of the tyrosines in lipid-free cytochrome b5 ionized immediately, whereas in the lipid-bound protein only 65% ionized within the first minute. In contrast, acetylation with acetylimidazole resulted in the conversion of all 5 tyrosine groups of lipid-free as well as lipid-bound cytochrome b5 into O-acetylated derivatives, which upon treatment with hydroxylamine were completely deacetylated. Reaction with N-bromosuccinimide revealed that only 60% of the 4 tryptophan residues present in cytochrome b5 were accessible to the reagent in the lipid-bound protein, although all tryptophans could be modified in lipid free cytochrome b5. It was concluded that the two tyrosines in the region linking the protein to the membrane are not shielded by lipid bilayer but that of the three tryptophans in the same region one is completely buried in the membrane, whereas the remaining two tryptophans may be both partly exposed to the solvent or alternatively, one may be partially and the other completely exposed.     

Age-dependent decay of cytochrome b5 and cytochrome b5 reductase in human erythrocytes.

Age-dependent decrease in cytochrome b5 was observed in erythrocytes from both a normal person and a patient with hereditary methaemoglobinaemia without neurological symptoms. With aging, concentrations of cytochrome b5 in erythrocytes from the patient were almost the same as those in the control. Age-dependent decrease in cytochrome b5 reductase activity in the control erythrocytes was also shown; however, the reductase activity was very low in erythrocytes from the patient over the whole age range. Our studies show that methaemoglobin content of erythrocytes seems to be dependent on the content of cytochrome b5 in the cells, both in the control subject and in the patient.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.