Biliverdin Reductase Is Heat Resistant and Coexpressed with Constitutive and Heat Shock Forms of Heme Oxygenase in Brain

Abstract: Two heme oxygenase (HO) isozymes—HO-1, which is a heat shock protein (HSP32), and HO-2—catalyze the isomer-specific production of biliverdin IXα and carbon monoxide. The latter has the potential of functioning as a neurotransmitter, whereas the reduced form of biliverdin, bilirubin, has potent antioxidant activity. Formation of bilirubin is catalyzed by biliverdin reductase (BVR). The reductase is a unique enzyme in being dual pyridine nucleotide and dual pH dependent. Here, we show that the reductase is resistant to thermal stress at both the protein and message level. We further demonstrate that the reductase is coexpressed in cells that display HO-1 and/or HO-2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock-inducible HO-1 protein. Exposure of male rats to 42°C for 20 min did not decrease brain BVR activity, but caused a slight increase in NADPH-and NADH-dependent activities at 1 and 6 h following hyperthermia. High levels of the ～ 1.5-kb BVR mRNA were detected in control brain; it too displayed thermal tolerance. Similarly, the pattern of multiplicity of net charge variants of the enzyme purified from brain of heat-shocked rats did not differ from the control pattern. Immunochemical localization of BVR protein in normal brain correlated well with the presence of HO-1 and/or HO-2 throughout the forebrain, diencephalon, cerebellum, and brainstem regions. There were select neuronal and nonneuronal cells in the substantia nigra and cerebellum that did express the reductase under normal conditions, wherein no HO isozymes could be detected. The same population, however, responded to heat shock by an intense increase in the level of HO-1. We postulate that the constitutive presence of the reductase in this cell population and the overall thermal stability of the enzyme represent a safeguard mechanism in the brain for the prompt conversion of biliverdin to bilirubin under conditions when oxidation of the heme moiety of denatured hemoproteins by HO-1 is accelerated.     

Protein/Protein Interactions in the Mammalian Heme Degradation Pathway: HEME OXYGENASE-2, CYTOCHROME P450 REDUCTASE,AND BILIVERDIN REDUCTASE*

Heme oxygenase (HO) catalyzes the rate-limiting step in the O₂-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The ¹H-¹⁵N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in K_m values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2·CPR complex (K_d = 15.1 μm). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the ¹H-¹⁵N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence.     

Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation

Biliverdin reductase (BVR) reduces heme oxygenase (HO) activity product, biliverdin, to bilirubin. BVR is unique in having dual pH/dual cofactor requirements. Using Escherichia coli-expressed human BVR and COS cells, we show that BVR is autophosphorylated and that phosphorylation is required for its activity. An "in blot" autophosphorylation assay showed that BVR is a renaturable phosphoprotein. Controls for the experiments were HO-1 and HO-2; both are phosphoproteins but are not autophosphorylated. Autophosphorylation was pH-dependent, with activity at pH 8.7 being most prominent. In addition, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate fluorescence titration of BVR gave a lower K(d) at pH 8.7 than at pH 7.4 (15.5 versus 28.0 micrometer). Mn(2+) was required for binding of the ATP analogue and for autophosphorylation; the autokinase activity was lost when treated at 60 degrees C for 10 min. The loss of transferred phosphates by alkaline treatment suggested that BVR is a serine/threonine kinase. Potato acid phosphatase treatment reversibly inactivated the enzyme. The enzyme was also inactivated by treatment with the serine/threonine phosphatase, protein phosphatase 2A; okadaic acid attenuated the inhibition. Titration of protein phosphatase 2A-released phosphates indicated a 1:6 molar ratio of BVR to phosphate. The BVR immunoprecipitated from COS cell lysates was a phosphoprotein, and its activity and phosphorylation levels increased in response to H(2)O(2). The results define a previously unknown mechanism for regulation of BVR activity and are discussed in the context of their relevance to heme metabolism.     

Human biliverdin reductase is a leucine zipper-like DNA-binding protein and functions in transcriptional activation of heme oxygenase-1 by oxidative stress.

Human biliverdin reductase (hBVR) is a serine/threonine kinase that catalyzes reduction of the heme oxygenase (HO) activity product, biliverdin, to bilirubin. A domain of biliverdin reductase (BVR) has primary structural features that resemble leucine zipper proteins. A heptad repeat of five leucines (L(1)--L(5)), a basic domain, and a conserved alanine characterize the domain. In hBVR, a lysine replaces L(3). The secondary structure model of hBVR predicts an alpha-helix-turn-beta-sheet for this domain. hBVR translated by the rabbit reticulocyte lysate system appears on a nondenaturing gel as a single band with molecular mass of approximately 69 kDa. The protein on a denaturing gel separates into two anti-hBVR immunoreactive proteins of approximately 39.9 + 34.6 kDa. The dimeric form, but not purified hBVR, binds to a 100-mer DNA fragment corresponding to the mouse HO-1 (hsp32) promoter region encompassing two activator protein (AP-1) sites. The specificity of DNA binding is suggested by the following: (a) hBVR does not bind to the same DNA fragment with one or zero AP-1 sites; (b) a 56-bp random DNA with one AP-1 site does not form a complex with hBVR; (c) in vitro translated HO-1 does not interact with the 100-mer DNA fragment with two AP-1 sites; (d) mutation of Lys(143), Leu(150), or Leu(157) blocks both the formation of the approximately 69-kDa specimens and hBVR DNA complex formation; and (e) purified preparations of hBVR or hHO-1 do not bind to DNA with two AP-1 sites. The potential significance of the AP-1 binding is suggested by the finding that the response of HO-1, in COS cells stably transfected with antisense hBVR, with 66% reduced BVR activity, to superoxide anion (O(2)()) formed by menadione is attenuated, whereas induction by heme is not affected. We propose a role for BVR in the signaling cascade for AP-1 complex activation necessary for HO-1 oxidative stress response.     

Prior induction of heme oxygenase-1 with glutathione depletor ameliorates the renal ischemia and reperfusion injury in the rat

Heme oxygenase (HO)-1 catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide, and biliverdin. Induction of HO-1 occurs as an adaptive and protective response to oxidative stress. Ischemia and reperfusion (IR) injury seems to be mainly caused by the oxidative stress. In this study, we have examined whether prior induction of HO-1 with buthionine sulfoximine (BSO), a glutathione (GSH) depletor, affects the subsequent renal IR injury. BSO (2 mmol/kg body weight) was administered intraperitoneally into rats, the levels of HO-1 protein increased within 4 h after the injection. When BSO was administered into rats at 5 h prior to the renal 45 min of ischemia, the renal IR injury was assessed by determining the levels of blood urea nitrogen and serum creatinine, markers for renal injury, after 24 h of reperfusion. The renal injury was significantly improved as compared to the rats treated with IR alone. Administration of zinc-protoporphyrin IX, an inhibitor of HO activity, reduced the efficacy of BSO pretreatment on the renal IR injury. Our findings suggest that the prior induction of HO-1 ameliorates the subsequent renal IR injury.     

Heme oxygenase-1 is upregulated in the rat heart in response to chronic administration of angiotensin II

Heme oxygenase (HO) is a heme-catabolizing enzyme that converts heme into biliverdin, iron, and carbon monoxide. HO-1, an inducible form of HO, is thought to act as an endogenous antioxidant defense mechanism. To determine whether chronic administration of angiotensin II affects HO-1 expression in the heart, expression and localization of HO-1 were investigated in the heart of rats receiving angiotensin II infusion (0.7 mg. kg(-1). day(-1)) via osmotic minipump for up to 7 days. Angiotensin II induced formation of granulation tissue, characterized by myofibroblast proliferation, fibrous deposition, and inflammatory cell migration. Angiotensin II also upregulated cardiac HO-1 expression. Immunohistochemistry revealed that HO-1 was intensively expressed in the granulation tissue. The selective AT(1)-receptor antagonist, losartan, completely, but hydralazine only partially, suppressed angiotensin II-induced granulation tissue formation and HO-1 upregulation. Chronic norepinephrine infusion (2.8 mg. kg(-1). day(-1)) did not induce granulation tissue formation or HO-1 upregulation. Our data suggest that angiotensin II upregulates cardiac HO-1 expression in the newly formed inflammatory lesion, which may represent an adaptive response to angiotensin II-induced cardiac damage.     

Immunohistochemical localization of the antioxidant enzymes biliverdin reductase and heme oxygenase-2 in human and pig gastric fundus

The intrinsic antioxidant capacities of the bile pigments biliverdin and bilirubin are increasingly recognized since both heme degradation products can exert beneficial cytoprotective effects due to their scavenging of oxygen free radicals and interaction with antioxidant vitamins. Several studies have been published on the localization of the carbon monoxide producing enzyme heme oxygenase-2 (HO-2), which concomitantly generates biliverdin; histochemical data on the distribution of biliverdin reductase (BVR), converting biliverdin to bilirubin, are still very scarce in large mammals including humans. The present study revealed by means of immunohistochemistry the presence of BVR and HO-2 in mucosal epithelial cells and in the endothelium of intramural vessels of both human and porcine gastric fundus. In addition, co-labeling with the specific neural marker protein-gene product 9.5 (PGP 9.5) demonstrated that both BVR and HO-2 were present in all intrinsic nerve cell bodies of both submucous and myenteric plexuses, while double labeling with c-Kit antibody confirmed their presence in intramuscular interstitial cells of Cajal (ICC). Our results substantiate the hypothesis that BVR, through the production of the potent antioxidant bilirubin, might be an essential component of normal physiologic gastrointestinal defense in man and pig.     

Heme-oxygenase-mediated iron accumulation in the liver

Heme oxygenase (HO) isozymes, HO-1 and HO-2, catalyze the conversion of heme to iron, carbon monoxide, and biliverdin. The present study was aimed at elucidating the role of the HO system in iron accumulation and oxidative stress in the liver. We have also studied the regulation of an iron exporter, ferroportin-1 (FPN-1), as an adaptive response mechanism to increased iron levels. Sprague-Dawley rats were treated with HO inducer hemin or HO inhibitor tin-protoporphyrin IX (SnPPIX) for 1 month. A portion of liver tissues was subjected to RT-PCR for HO-1, HO-2, and FPN-1 gene expression as well as an HO activity assay. Paraffin-embedded tissues were stained for iron with Prussian blue. Hepatic iron concentration was measured by High Resolution-Inductively Coupled Plasma-Mass Spectrometry. 8-hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of oxidative DNA damage, was performed to assess oxidative stress. Hemin treatment led to augmented HO expression and activity in association with increased iron accumulation and oxidative stress. FPN-1 expression was also found to be upregulated. SnPPIX treatment reduced HO activity, intracellular iron levels, and oxidative stress as compared to controls. Our data provides evidence of increased HO activity as an important pro-oxidant mechanism leading to iron accumulation in the liver.     

Heme oxygenase-1 and the ischemia-reperfusion injury in the rat heart

Carbon monoxide (CO) is a signaling gas produced intracellularly by heme oxygenase (HO) enzymes using heme as a substrate. During heme breakdown, HO-1 and HO-2 release CO, biliverdin, and Fe(2+). In this study, we investigated the effects of manipulation of the HO-1 system in an in vivo model of focal ischemia-reperfusion (FIR) in the rat heart. Male Wistar albino rats, under general anesthesia and artificial ventilation, underwent thoracotomy, the pericardium was opened, and a silk suture was placed around the left descending coronary artery; ischemia was induced by tightening the suture and was monitored for 30 min. Subsequently, the ligature was released to allow reperfusion lasting for 60 min. The first group of rats was sham operated and injected intraperitoneally (i.p.) with saline. The second group underwent FIR. The third group was treated ip 18 hr before FIR with hemin (4 mg/kg). The fourth group was pretreated ip 24 hr before FIR and 6 hr before hemin with zinc protoporphyrin IX (ZnPP-IX, 50 microg/kg). Specimens of the left ventricle were taken for determination of HO expression and activity, infarct size, malonyldialdehyde (MDA) production, and tissue calcium content. FIR led to a significant increase in the generation of MDA and notably raised tissue calcium levels. Induction of HO-1 by hemin significantly decreased infarct size, incidence of reperfusion arrhythmias, MDA generation, and calcium overload induced by FIR. These effects were prevented by the HO-1 inhibitor ZnPP-IX. The present experiments show that the concerted actions of CO, iron, and biliverdin/bilirubin modulate the FIR-induced myocardial injury.     

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.     

Carbon monoxide and biliverdin prevent endothelial cell sloughing in rats with type I diabetes

Hyperglycemia has been linked to increased oxidative stress, a resultant endothelial cell dysfunction, and, ultimately, apoptosis. Heme oxygenases (HO-1/HO-2) and the products of their activity, biliverdin/bilirubin and carbon monoxide (CO), play a physiological role in the vascular system. The effects of heme-mediated HO-1 induction, CO, and biliverdin on urinary 8-epi-isoprostane PGF₂ and endothelial cell sloughing were examined in an animal model of streptozotocin (STZ)-induced diabetes. Hyperglycemia itself did not affect HO-1 and HO-2 protein levels, but caused a net decrease in HO activity. Weekly heme administration induced HO-1 protein, as demonstrated by immunohistochemistry and Western blot analyses. Administration of biliverdin or the CO donor, CORM-3, decreased urinary 8-epi-isoprostane PGF₂, P < 0.5 compared to diabetes. Hyperglycemia increased endothelial cell sloughing; 8.2 ± 0.8 cells/ml blood in control rats vs. 48 ± 4.8 cells/ml blood in diabetic rats (P < 0.05). Heme administration significantly increased endothelial cell sloughing in diabetic rats (98 ± 8.1 cells/ml blood, P < 0.0007) whereas biliverdin modestly decreased endothelial cell sloughing (26 ± 3.5 cells/ml blood, P < 0.003). Administration of CORM-3 to diabetic rats resulted in a significant decrease in endothelial cell sloughing to 21.3 ± 2.3 (P < 0.001). Administration of SnMP to CORM-3 diabetic rats only partially reversed the protective effects of CORM-3 on endothelial cell sloughing from 21.3 ± 2.3 to 29 ± 2.1 cells/ml, thus confirming a direct protective of CO, in addition to the ability of CORM-3 to induce HO-1 protein. These results demonstrate that exogenously administered CO or bilirubin can prevent endothelial cell sloughing in diabetic rats, likely via a decrease in oxidative stress, and thus represents a novel approach to prophylactic vascular protection in diabetes.     

Differential effect of cobalt protoporphyrin on distributions of heme oxygenase in renal structure and on blood pressure in SHR.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, releasing iron and carbon monoxide (CO). Thus, HO not only controls the availability of heme for the synthesis of hemeproteins but also generates CO, which binds to the heme moiety of hemoproteins, thereby affecting their enzymatic activity. The present study was undertaken to explore changes in the relative expression of renal HO-1 and HO-2 in response to modulators and the effect on blood pressure regulation in spontaneously hypertensive rats (SHR). Immunohistochemistry confirmed a cobalt protoporphyrin (CoPP)-mediated increase in HO-1 protein. After a single injection of CoPP (5 mg/100 gram body weight) in 7-week-old SHR, blood pressure significantly decreased (p<0.01) while renal HO activity increased 6-fold over controls. CoPP pretreatment deceased the levels of the renal cytochrome P450-derived arachidonic acid metabolite, 20-HETE, a powerful vasoconstrictor, by 65% in renal tissue. Western blot analysis demonstrated that CoPP significantly increased HO-1 protein expression in the cortex and outer medulla and, to a lesser degree, in the inner medulla of the rat kidney. HO-2 was constitutively expressed in all parts of the kidney, and did not significantly change after treatment with CoPP. These results indicate that selective induction of cortical and outer medullary HO-1 is associated with a decrease in 20-HETE and blood pressure, suggesting an important role for HO-1 activity in the regulation of urine volume, electrolyte excretion and blood pressure.     

Protective role of heme oxygenase in the blood vessel wall during atherogenesis.

Several lines of evidence suggest that antioxidant processes and (or) endogenous antioxidants inhibit proatherogenic events in the blood vessel wall. Heme oxygenase (HO), which catabolizes heme to biliverdin, carbon monoxide, and catalytic iron, has been shown to have such antioxidative properties. The HO-1 isoform of heme oxygenase is ubiquitous and can be increased several fold by stimuli that induce cellular oxidative stress. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a role in modulation of vascular tone; biliverdin and its by-product bilirubin are potent antioxidants. Although HO induction results in an increase in catalytic free iron release, the enhancement of intracellular ferritin protein through HO-1 has been reported to decrease the cytotoxic effects of iron. Oxidized LDL has been shown to increase HO-1 expression in endothelial and smooth muscle cell cultures, and during atherogenesis. Further evidence of HO-1 expression associated with atherogenesis has been demonstrated in human, murine and rabbit atherosclerotic lesions. Moreover, genetic models of HO deficiency suggest that the actions of HO-1 are important in modulating the severity of atherosclerosis. Recent experiments in gene therapy using the HO gene suggest that interventions aimed at HO in the vessel wall could provide a novel therapeutic approach for the treatment or prevention of atherosclerotic disease.     

Heme oxygenase-1 posttranslational modifications in the brain of subjects with Alzheimer disease and mild cognitive impairment

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Oxidative and nitrosative stress plays a principal role in the pathogenesis of AD. The induction of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species. Although initially proposed as a neuroprotective system in AD brain, the HO-1/BVR-A pathophysiological features are under debate. We previously reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative posttranslational modifications in the brain of subjects with AD and those with mild cognitive impairment (MCI). Furthermore, other groups proposed the observed increase in HO-1 in AD brain as a possible neurotoxic mechanism. Here we provide new insights about HO-1 in the brain of subjects with AD and MCI, the latter condition being the transitional phase between normal aging and early AD. HO-1 protein levels were significantly increased in the hippocampus of AD subjects, whereas HO-2 protein levels were significantly decreased in both AD and MCI hippocampi. In addition, significant increases in Ser-residue phosphorylation together with increased oxidative posttranslational modifications were found in the hippocampus of AD subjects. Interestingly, despite the lack of oxidative stress-induced AD neuropathology in cerebellum, HO-1 demonstrated increased Ser-residue phosphorylation and oxidative posttranslational modifications in this brain area, suggesting HO-1 as a target of oxidative damage even in the cerebellum. The significance of these findings is profound and opens new avenues into the comprehension of the role of HO-1 in the pathogenesis of AD.     

Inhibition of heme oxygenase augments tubular sodium reabsorption

Heme oxygenase (HO) catalyzes the degradation of heme to form iron, biliverdin, and carbon monoxide (CO). The vascular actions of CO include direct vasodilation of vascular smooth muscle and indirect vasoconstriction through inhibition of nitric oxide synthase (NOS). This study was performed to examine the effects in the kidney of inhibition of heme oxygenase alone or combined with NOS inhibition. Chromium mesoporphyrin (CrMP; 45 μmol/kg ip), a photostable HO inhibitor, was given to control rats and N(G)-nitro-l-arginine methyl ester (l-NAME)-treated hypertensive rats (50 mg·kg?1·day?1), 12 h, 4 days). In control animals, CrMP decreased CO levels, renal HO-1 levels, urine volume, and sodium excretion, but had no effect on arterial pressure, renal blood flow (RBF), plasma renin activity (PRA), or glomerular filtration rate (GFR). In l-NAME-treated hypertensive rats, CrMP decreased endogenous CO and renal HO-1 levels and had no effect on arterial pressure, RBF, or GFR but decreased sodium and water excretion in a similar manner to control animals. An increase in PRA was observed in untreated rats but not in l-NAME-infused rats, indicating that this effect is associated with an absent NO system. The results suggest that inhibition of HO promotes water and sodium excretion by a direct tubular action that is independent of renal hemodynamics or the NO system.     

Heme oxygenase metabolites inhibit tubuloglomerular feedback in vivo

Tubuloglomerular feedback (TGF) is a renal autoregulatory mechanism that constricts the afferent arteriole in response to increases in distal NaCl. Heme oxygenases (HO-1 and HO-2) release carbon monoxide (CO) and biliverdin, which may help control renal function. We showed in vitro that HO products inhibit TGF; however, we do not know whether this also occurs in vivo or the mechanism(s) involved. We hypothesized that in vivo HO-1 and HO-2 in the nephron inhibit TGF via release of CO and biliverdin. We first performed laser capture microdissection followed by real-time PCR and found that both HO-1 and HO-2 are expressed in the macula densa. We next performed micropuncture experiments in vivo on individual rat nephrons, adding different compounds to the perfusate, and found that an HO inhibitor, stannous mesoporphyrin (SnMP), potentiated TGF (P < 0.05, SnMP vs. control). The CO-releasing molecule (CORM)-3 partially inhibited TGF at 50 μmol/l (P < 0.01, CORM-3 vs. control) and blocked it completely at higher doses. A soluble guanylyl cyclase (sGC) inhibitor, LY83583, blocked the inhibitory effect of CORM-3 on TGF. Biliverdin also partially inhibited TGF (P < 0.01, biliverdin vs. control), most likely attributable to decreased superoxide (O(2)(-)) because biliverdin was rendered ineffective by tempol, a O(2)(-) dismutase mimetic. We concluded that HO-1 and HO-2 in the nephron inhibit TGF by releasing CO and biliverdin. The inhibitory effect of CO on TGF is mediated by the sGC/cGMP signaling pathway, whereas biliverdin probably acts by reducing O(2)(-).     

Multiple forms of biliverdin reductase: modification of the pattern of expression in rat liver by bromobenzene

Rat liver biliverdin reductase was purified from control and bromobenzene-treated rats and was designated as C-BVR-T and B-BVR-T, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the existence of two molecular weight variants (30,100 and 29,800) in C-BVR-T but only one form (30,100) in B-BVR-T. Western immunoblotting confirmed that both molecular weight variants were biliverdin reductase. Nondenaturing electrophoresis separated C-BVR-T and B-BVR-T preparations into groups of four variants, designated as BVR ND1 to ND4. However, the C-BVR-T preparation contained three major forms (BVR ND1, ND2, and ND3) while the B-BVR-T preparation contained two major forms (BVR ND2 and ND3). In vitro treatment of biliverdin reductase preparations with either bromobenzene or dithiothreitol did not interconvert the variants of the enzyme. QAE-Sepharose anion-exchange chromatography was used to isolate the ND2 and ND3 variants for physiochemical analysis. The amino acid composition of the variants was rather similar except for their Tyr content. Also, the peptide maps were similar except for a series of moderately early chromatographic peaks. These findings implied secondary modifications to the protein rather than substantial differences in primary structure. The pH-dependent cofactor requirements for enzyme activity were examined. Both variants exhibited 2 pH optima that were cofactor dependent; maximum activity with NADPH and NADH was observed at pH 8.5 and 6.7, respectively. However, both variants exhibited a higher catalytic rate with NADH than with NADPH at their pH optima. Furthermore, BVR ND3 exhibited a higher catalytic rate than BVR ND2 with either cofactor throughout the pH range 6.5-9.