Reduction of Nω-hydroxy-L-arginine by the mitochondrial amidoxime reducing component (mARC)

Abstract

NOSs (nitric oxide synthases) catalyse the oxidation of L-arginine to L-citrulline and nitric oxide via the intermediate NOHA (N(ω)-hydroxy-L-arginine). This intermediate is rapidly converted further, but to a small extent can also be liberated from the active site of NOSs and act as a transportable precursor of nitric oxide or potent physiological inhibitor of arginases. Thus its formation is of enormous importance for the nitric-oxide-generating system. It has also been shown that NOHA is reduced by microsomes and mitochondria to L-arginine. In the present study, we show for the first time that both human isoforms of the newly identified mARC (mitochondrial amidoxime reducing component) enhance the rate of reduction of NOHA, in the presence of NADH cytochrome b₅ reductase and cytochrome b₅, by more than 500-fold. Consequently, these results provide the first hints that mARC might be involved in mitochondrial NOHA reduction and could be of physiological significance in affecting endogenous nitric oxide levels. Possibly, this reduction represents another regulative mechanism in the complex regulation of nitric oxide biosynthesis, considering a mitochondrial NOS has been identified. Moreover, this reduction is not restricted to NOHA since the analogous arginase inhibitor NHAM (N(ω)-hydroxy-N(δ)-methyl-L-arginine) is also reduced by this system.

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

“…With the exception of the human Moco sulfurase HMCS, which is required for activating the Mo-enzymes AO and XOR (9) and which is the name-giving member of the MOSC family, no other homolog of hmARC-1 and hmARC-2 is found in the human genome. Cloning of hmARC-1 and hmARC-2 cDNAs derived from HepG2 cells (16,17) basically confirmed the predicted sequences of the protein-encoding open reading frames as deposited in the database, with the exception of two reproducible polymorphisms that were identified in the hmARC-1 cDNA causing substitutions of methionine 187 by lysine (protein entry NP_073583 versus AAH10619) and alanine 165 by threonine (protein entry ACB21046 versus NP_073583).…”

“…Construction of Expression Vectors-Full-length cDNAs of hmARC-1 (1011 base pairs corresponding to NCBI reference sequence NM_022746, MOSC-1) and hmARC-2 (1005 base pairs corresponding to NCBI reference sequence NM_017898, MOSC-2) were obtained as described earlier (15)(16)(17). For cloning of N-terminally truncated hmARC-1, the full-length open reading frame was truncated at its 5Ј end by PCR using the primers hmARC-1_Start_N-del (5Ј-ATA TAT GGA TCC ATG CAG CAG GTG GGC ACA GTG GCG-3Ј) and hmARC1_Stop (5Ј-AAA TTT AAG CTT TTA CTG GCC CAG CAG GTA CAC AGG-3Ј).…”

“…Determination of the N-Reductive Activity of hmARC-1 and hmARC-2-N-Reductive activities of hmARC-1 and hmARC-2 with benzamidoxime, N-hydroxy-L-arginine, or N-hydroxysulfonamides as substrates were determined as described previously with minor modifications (16,17). In the case of benzamidoxime, either 100 mM potassium phosphate buffer, pH 6.0 (for experiments shown in Figs.…”

“…Thus, not only the cofactor composition but also the order of cofactors and the direction of electron flow are identical to NR. However, although many N-hydroxylated compounds have been found to serve as substrates for native and recombinant mARC proteins (15)(16)(17), the physiological substrates, and thus the physiological function, of mARC proteins are as yet unknown. The present study aimed to provide the first detailed biochemical information about the heterologously expressed human mARC isoforms hmARC-1 and hmARC-2 with particular focus on the characterization of their molybdenum center.…”

The Pivotal Role of the Mitochondrial Amidoxime Reducing Component 2 in Protecting Human Cells against Apoptotic Effects of the Base Analog N6-Hydroxylaminopurine

“…Besides its involvement in metabolic detoxication other functions of mARC have been proposed. mARC has been suggested to be involved in NO metabolism with regulatory functions in the L-arginine-dependent biosynthesis of NO (28) or reduction of nitrite to NO under anaerobic conditions (29). Furthermore, mARC is brought into context with energy and lipid metabolism (30 -32) as well as with metabolic disorders as diabetes mellitus (33).…”