The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells

Abstract

The mitochondrial amidoxime reducing component mARC is a recently discovered molybdenum enzyme in mammals. mARC is not active as a standalone protein, but together with the electron transport proteins NADH-cytochrome b5 reductase (CYB5R) and cytochrome b5 (CYB5), it catalyzes the reduction of N-hydroxylated compounds such as amidoximes. The mARC-containing enzyme system is therefore considered to be responsible for the activation of amidoxime prodrugs. All hitherto analyzed mammalian genomes code for two mARC genes (also referred to as MOSC1 and MOSC2), which share high sequence similarities. By RNAi experiments in two different human cell lines, we demonstrate for the first time that both mARC proteins are capable of reducing N-hydroxylated substrates in cell metabolism. The extent of involvement is highly dependent on the expression level of the particular mARC protein. Furthermore, the mitochondrial isoform of CYB5 (CYB5B) is clearly identified as an essential component of the mARC-containing N-reductase system in human cells. The participation of the microsomal isoform (CYB5A) in N-reduction could be excluded by siRNA-mediated down-regulation in HEK-293 cells and knock-out in mice. Using heme-free apo-CYB5, the contribution of mitochondrial CYB5 to N-reductive catalysis was proven to strictly depend on heme. Finally, we created recombinant CYB5B variants corresponding to four nonsynonymous single nucleotide polymorphisms (SNPs). Investigated mutations of the heme protein seemed to have no significant impact on N-reductive activity of the reconstituted enzyme system.

Keywords: CYB5B; Drug Metabolism; Heme; MOSC; Mitochondria; Molybdenum; N-reduction; RNA Interference (RNAi); mARC.

FIGURE 1.

N-Reductive enzyme system in human cells.

FIGURE 2.

mARC1 and mARC2 knockdown in human cells. A, mARC1 knockdown in HEK-293. B, mARC2 knockdown in HEK-293. C, mARC1 and mARC2 knockdown in ZR-75-1. For HEK-293, cells were transfected with various amounts of mARC1, mARC2, or nontargeting (NC) siRNA. For ZR-75-1, cells were transfected with 20 nm of NC, mARC1, or mARC1 siRNA. For simultaneous knockdown, 10 nm mARC1 and 10 nm mARC2 siRNA were transfected. N-Reductive activities in cells were determined as described under “Experimental Procedures” on day 5 (for HEK-293) or day 6 (for ZR-75-1) after transfection. Results are presented as means ± S.D. (n = 3). **, p < 0.01, ***, p < 0.001. The siRNA-mediated down-regulations of the proteins of interest were verified by Western blot using anti-mARC1, anti-mARC2, or anti-calnexin (loading control) antibody.

FIGURE 3.

CYB5A and CYB5B knockdown in HEK-293 cells. A, CYB5A knockdown. B, CYB5B knockdown. Cells were transfected with various amounts of CYB5A, CYB5B, or nontargeting (NC) siRNA. N-Reductive activities in HEK-293 cells were determined as described under “Experimental Procedures” on day 5 after transfection. Results are presented as means ± S.D. (n = 3). *, p < 0.05, ***, p < 0.001. The siRNA-mediated down-regulations of the proteins of interest were verified by Western blot using anti-CYB5A, anti-CYB5B, or anti-GAPDH (loading control) antibody.

FIGURE 4.

N-Reductive activities of subcellular liver fractions of CYB5A knock-out mice. N-Reductive activities of subcellular fractions of wild type and CYB5A knock-out mouse livers were determined as described under “Experimental Procedures.” Assays were performed on a pool of two liver preparations and one individual liver sample of wild type or knock-out mice (n = 3). Incubations were performed in duplicates. Results are presented as means ± S.D. The absence of CYB5A protein in the individual knock-out samples was verified by Western blot analysis using anti-CYB5A antibody.

FIGURE 5.

Reduction of benzamidoxime using cytochrome b₅ type B wild type and variants. A, assay carried out with mARC1. B, assay carried out with mARC2. 120 pmol of molybdenum (mARC), 30–210 pmol of heme (CYB5B), and 6 pmol of FAD (CYB5R) were incubated with 3 mm benzamidoxime in 20 mm MES, pH 6.0, for 15 min under aerobic conditions. Data are means ± S.D. of one batch (CYB5B variants) or of four batches (CYB5B WT) each incubated in duplicates and measured twice by HPLC.

FIGURE 6.

Involvement of cytochrome b₅ heme in N-reduction. Recombinant CYB5 (holo-CYB5) was gained as described under “Experimental Procedures.” Apo-CYB5 was prepared from holo-CYB5 by heme cleavage, and afterward the same batch was reconstituted by heme incorporation. The human recombinant N-reductive enzyme system was then reconstituted with mARC1, CYB5R, and either form of CYB5. N-Reductive activities were measured with benzamidoxime as a model substrate. A, heme saturations of holo-CYB5, apo-CYB5, and reconstituted CYB5 were determined to confirm the success of heme cleavage and heme incorporation. B, N-reductive activities of holo-CYB5, apo-CYB5, and reconstituted CYB5 were determined as described under “Experimental Procedures.” The incubation mix contained 75 pmol of mARC, 7.5 pmol of CYB5R, and 75 pmol of CYB5 in the case of holo and reconstituted CYB5 or the same amount of protein as holo-CYB5 in the case of apo-CYB5. Incubations were performed in duplicates. Results are presented as means ± S.D.