Redox properties of the lipocalin α1-microglobulin: Reduction of cytochrome c, hemoglobin, and free iron

Abstract

Abstractα 1 -microglobulin (α 1 m) is a 26 kDa plasma and tissue glycoprotein. The protein has a heterogeneous yellow-brown chromophore consisting of small unidentified prosthetic groups localized to a free thiol group (C34) and three lysyl residues (K92, K118 and K130) around the entrance to a hydrophobic pocket. It was recently reported that α 1 m can bind heme and that a C-terminally processed form of α 1 m degrades heme. It is shown here that α 1 m has catalytic reductase and NADH-dehydrogenase-like activities. Cytochrome c, nitroblue tetrazolium (NBT), methemoglobin and ferricyanide were reduced by α 1 m. Comparison of the reduction rates suggests that methemoglobin is a better substrate than cytochrome c, NBT and ferricyanide. The reactions with cytochrome c and NBT were mediated by superoxide anions since they were inhibited by superoxide dismutase. The addition of the biological electron donors NADH, NADPH or ascorbate enhanced the reduction rate of cytochrome c approximately 30-fold. Recombinant α 1 m, which has much less chromophore than plasma and urine α 1 m, was a stronger reductant than the latter α 1 m-forms. Site-directed mutagenesis of C34, K92, K118 and K130 and thiol group chemistry showed that the C34 thiol group was involved in the redox reaction but relies upon co-operation with the lysyl residues. The redox properties of α 1 m may provide a physiological protection mechanism against extracellularly exposed heme-groups and other oxidants.

A1M, an extravascular tissue cleaning and housekeeping protein

“…Enzymatic reductase/dehydrogenase properties were recently described for A1M with a wide spectrum of organic and inorganic substrates ( Figure 3A) [10,38]. The protein was capable of reducing the heme proteins cytochrome c and methemoglobin, free iron and the synthetic compounds nitroblue tetrazolium (NBT) [38].…”

“…The protein was capable of reducing the heme proteins cytochrome c and methemoglobin, free iron and the synthetic compounds nitroblue tetrazolium (NBT) [38]. The results thus suggest that A1M is rather unspecific with regard to its substrates.…”

“…The results thus suggest that A1M is rather unspecific with regard to its substrates. A catalytic reductase effect was seen in the presence of ascorbate, NADH and NADPH [38], but these are probably not the only physiologically relevant electron-donating co-factors, since the reducing activity of A1M has been shown both intra-and extracellularly (see below). The reductase and dehydrogenase activities are dependent on the thiol group of Cys34 and the three lysyl residues of Lys92, 118 and 130.…”

“…The three Lys residues were proposed to function by creating a positively charged microenvironment around the Cys34, thus lowering the pK a of the thiol group and favoring its oxidation [38]. In addition, A1M rapidly reduced the synthetic radical 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and this reaction was also dependent on the free thiol group of Cys34 [10].…”

“…Furthermore, the tryptophan metabolite 3-OH-kynurenin, which has a propensity to form free radicals [48], was identified as a lysyl modification on urinary A1M from hemodialysis patients [49]. The side-chains of Lys92, 118 and 130 may therefore serve a dual role both in activation of the Cys34 thiol by lowering its pK a [38] and as electron-donors in the radical-trapping mechanism. Finally, it has also been reported that the Cys34 thiol itself may be covalently modified by unidentified brown-colored oxidation products in vivo [50].…”