Conversion of cytochrome c into a peroxidase: Inhibitory mechanisms and implication for neurodegenerative diseases

Abstract

a b s t r a c tA further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H 2 O 2 . To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.

The Met80Ala and Tyr67His/Met80Ala mutants of human cytochrome c shed light on the reciprocal role of Met80 and Tyr67 in regulating ligand access into the heme pocket

“…) [13], in which the heme Met80 axial ligand bond is ruptured, is lower than that reported here for the MA variant. On the other hand, the apparent k cat /K m value for the human YHMA mutant is only marginally increased with respect to that of the human WT and Y67H mutant [14].…”

“…At pH 7.0 and 25°C, WT human cyt c exhibits a very low peroxidase activity, with an apparent performance constant (k cat /K m ) comparable to that of WT horse heart cyt c [13]. The MA variant, where cleavage of the Fe-Met80 bond occurs, displays more favorable apparent k cat /K m values than WT for peroxidation of guaiacol (see Table 1).…”

“…The procedures are outlined briefly in the Supplementary information together with the primer sequences used in this work (Table S1). Purification of the recombinant proteins was conducted as previously described [13]. After purification the proteins were concentrated and stored in 0.1 M phosphate buffer at pH 7.0.…”

“…The CO complexes were prepared by degassing the ferric protein solution by flushing firstly with nitrogen, then with 12 CO or 13 CO and reducing the heme by addition of 2-3 μL of a freshly prepared sodium dithionite (10 mg/mL) solution.…”

“…Moreover, mutations at the Tyr67 position have been previously shown to lead to a rearrangement of the distal portion of the heme cavity, depending on the type of substituting residue [11,12]. In human cyt c, substitution of Tyr67 by Arg leads to the cleavage of the Met80 heme bond and to an increased peroxidase activity, while substitution by His affects the heme pocket to a much lower extent and the Tyr67His mutant seems to have no significant peroxidase activity [13,14]. Strikingly, in yeast cyt c, the Tyr67His mutant has been reported to have significant peroxidase activity, possibly acting as a distal histidine and general acid-base catalyst in the distal heme pocket [15].…”