Human heme oxygenase-1 efficiently catabolizes heme in the absence of biliverdin reductase

Abstract

Heme oxygenase 1 (HO-1) uses molecular oxygen and electrons from NADPH cytochrome P450 reductase to convert heme to CO, ferrous iron, and biliverdin (BV). Enzymatic studies with the purified 30-kDa form of HO-1 routinely use a coupled assay containing biliverdin reductase (BVR), which converts BV to bilirubin (BR). BVR is believed to be required for optimal HO-1 activity. The goal of this study was to determine whether HO-1 activity could be monitored directly by following BV generation or iron release (using the ferrous iron chelator, ferrozine) in the absence of BVR. Using assays for each of the three end products, we found that HO-1 activity was stimulated in the presence of catalase and comparable rates were measured with each assay. Absorbance scans revealed characteristic spectra for BR, BV, and/or the ferrozine-iron complex. The optimal conditions were slightly different for the direct and coupled assays. BSA activated the coupled but inhibited the direct assays, and the assays had different pH optima. By measuring the activity of BVR directly using BV as a substrate, these differences were attributed to different enzymatic properties of BVR and HO-1. Thus, BVR is not needed to measure the activity of HO-1 when catalase is present. In fact, the factors affecting catalysis by HO-1 are better understood using the direct assays because the coupled assay can be influenced by properties of BVR.

Fig. 1.

Schematic diagram describing the functional molecular interactions involved in sHO-1 catalysis. Top, sHO-1 reaction according to the widely accepted opinion that BVR binding causes the release of BV from the sHO-1 active site. The binding of proteins to sHO-1 are indicated by the black double-headed arrows. The binding of CPR (1) and BVR (2) must occur in the specified sequence to complete the catalytic cycle. The reaction arrows (curved, gray) indicate the products released/formed during each of the protein-binding events. This sequence of events implies that a complicated series of protein-protein interactions is required for HO-1 catalysis. Bottom, a reaction sequence (supported by the results in this study) in which BVR does not need to bind to the sHO-1 for optimal catalysis. As a result, the panel shows that only one binding event is required between the sHO-1 and the CPR to complete the sHO-1 catalytic cycle. Also shown in both panels is an arrow representing a putative binding event between P450 and sHO-1.

Fig. 2.

Comparison of sHO-1 activity by direct detection of BV and Fe-ferrozine and the coupled assay measuring BR formation: effect of catalase. All reactions contained 0.1 μM sHO-1, 0.3 μM CPR, and 15 μM heme in 100 mM MOPS buffer (pH 7.4), either in the absence or presence of 0.25 unit/μl catalase. All reactions were incubated at 37°C and were initiated by the addition of 0.4 mM NADPH. A, HO-1 activity was measured using a coupled assay containing 0.2 μM BVR as described under Materials and Methods. Formation of BR was monitored at 468 nm. B, when the Fe-ferrozine complex was used to measure HO-1 activity, the assay included 0.25 mM ferrozine in addition to the enzymes heme and NADPH. The ferrozine complex was monitored at 564 nm. C, BV formation was measured directly using its absorbance (670 nm). The dotted line represents an estimate of the initial rate of the reaction as determined by a fit through the linear portion of the data.

Fig. 3.

Absorbance spectra of the various complexes used for detection of sHO-1 activity. The spectra were taken 300 s after addition of NADPH for each of the reactions described in Fig. 1. The absorbances of “reference” solutions containing all of the reaction constituents except NADPH were subtracted from those of the reaction incubations. The curves represent BV (dotted line), ferrozine (solid line), and BR (dashed line).

Fig. 4.

Purity of three preparations of commercially available catalase. Coomassie stain of polyacrylamide gel after electrophoresis of bovine liver catalase purchased from USB (lanes 1 and 5), EMD Biosciences (lanes 2 and 6), and Sigma-Aldrich (lanes 3 and 7). Fifteen microliters of each batch was loaded on the gel at 10 and 3.3 units/μl concentrations. Lane 4 shows 15 μl of a protein ladder (MagicMark from Invitrogen, Carlsbad, CA) molecular weight standard.

Fig. 5.

Effect of buffer on sHO-1 activity. Activity of sHO-1 was measured directly by BV formation and iron release and indirectly by BR formation in 0.1 M MOPS, 0.1 M potassium phosphate (KPi), and 0.1 M Tris buffers at pH 7.4. Assays for the production of BV, BR, and the iron-ferrozine complex are described under Materials and Methods. The data represent the average ± S.E. of nine determinations in three experiments. Statistical significance was determined by Dunnett's analysis of variance test for MOPS and Tris data using the activity as determined by the direct detection of BV as a control and a Student's t test using the Welch correction for the potassium phosphate data (*, p < 0.05; **, p < 0.01). N.D., not detected.

Fig. 6.

Direct comparison of the assays used to measure sHO-1 activity. Reactions containing sHO-1, CPR, heme, and catalase to measure the formation of BV, BR, and the iron-ferrozine complex were the same as those described under Materials and Methods except that 0.2, 0.4 (2× BVR), and 0.1 (1/2× BVR) μM BVR were added to the incubations labeled “bilirubin.” BV (biliv) formation was also measured in one group of samples in the presence of 0.25 mM ferrozine (ferroz), as indicated. The data represent the average ± S.E. of 12 determinations in four experiments. Statistical significance was determined using Dunnett's analysis of variance test, using the corresponding MOPS groups as a control (*, p < 0.05; **, p < 0.01).

Fig. 7.

Effect of incubation conditions on the rates of metabolism by sHO-1 and BVR. A, activity of sHO-1 was measured by the formation of BV, BR, and the iron-ferrozine complex as described under Materials and Methods. All reactions contained 100 mM MOPS (pH 7.4) and 0.25 unit/μl catalase and the following reaction components as indicated: 0.125 mg/ml BSA, 100 mM NaCl, and 20% glycerol. B, BVR activity was measured by monitoring the generation of BR from BV. The assay contained 0.02 μM BVR and 20 μM BV in 100 mM MOPS and the components indicated in the figure. The data represent the average ± S.D. of six determinations from two experiments. Statistical significance was determined using Dunnett's test, using the corresponding MOPS groups as a control (*, p < 0.05; **, p < 0.01).

Fig. 8.

Effect of pH on sHO-1 activity. Reactions were performed as described under Materials and Methods in 100 mM MOPS buffer at the pH values indicated. The coupled assays, which included 0.2 μM BVR, were performed in the presence of 0.25 mg/ml BSA. The data represent the average ± S.D. of six determinations collected in two experiments.