A novel role for cytochrome c: Efficient catalysis of S-nitrosothiol formation

Abstract

While S-nitrosothiols are regarded as important elements of many NO-dependent signal transduction pathways, the physiological mechanism of their formation remains elusive. Here, we demonstrate a novel mechanism by which cytochrome c may represent an efficient catalyst of S-nitrosation in vivo. In this mechanism, initial binding of GSH to ferric cytochrome c is followed by reaction of NO with this complex, yielding ferrous cytochrome c and GSNO. We show that when submitochondrial particles or cell lysates are exposed to NO in the presence of cytochrome c, there is a robust formation of protein S-nitrosothiols. In the case of submitochondrial particles protein S-nitrosation is paralleled with an inhibition of mitochondrial complex I. These observations raise the possibility that cytochrome c is a mediator of S-nitrosation in biological systems, particularly during hypoxia, and that release of cytochrome c in to the cytosol during apoptosis potentially releases a GSNO synthase activity which could modulate apoptotic signaling.

The Chemical Biology of S-Nitrosothiols

“…A fortunate exception to this is S-nitrosoglutathione (GSNO), which can be easily synthesized and isolated as a pure compound. Similar to tertiary S-nitrosothiols, such as (Ph) 3 CSNO and t-BuSNO, Snitroso-N-acetyl penicillamine (SNAP) is green in color and is one of the most commonly used S-nitrosothiols in biological applications (See Fig. 1 for the structures of S-nitrosothiols that are commonly used in biological experiments).…”

S-nitrosylation: integrator of cardiovascular performance and oxygen delivery

“…Similarly, transfer of metal-coordinated NO from cytochrome c (22) or ceruloplasmin (23) is employed in the synthesis of S-nitrosoglutathione (GSNO). Cys-to-Cys nitrosylases (referred to herein as transnitrosylases) are typically S-nitroso-proteins (SNO-proteins) involved in Cys-to-Cys transfer of the NO group from donor to acceptor protein (9,24).…”

Preservation of Nitric Oxide Availability as Nitrite and Nitrosothiols

“…In addition, the effects of transition metal ions [89], cellular antioxidants [90] and lipid radical species [91] and pH variations [74] need to be further examined. There is also a possibility of a heme enzyme dependent nitrosation [92]. Glutathione may bind to ferricytochrome c followed by a combination of NO to form a ferric cyt-c GSNOH complex.…”

“…In chapter 2 of this work, we showed that GSNO formation was negligible through the N 2 O 3 route. Consequently, GSNO formation was compared between two mechanisms: 1) a · NO 2 radical based nitrosation scheme as has been described in Chapter 2 of this work and 2) the process of ferricytochrome c reduction as has been described by Basu et al [151] and Broniowska et al [93]. According to the proposed mechanism for the GSNO steady state was achieved by assuming a GSNO half-life in blood of about 5.5…”

“…GSNO poduction through generation of the GS · radical by oxidation of GSH with · NO 2 with the kinetics described in chapter 3 of this work and through the process of ferricytochrome c reduction will be done [151], [93]. The ability of GSNO to deliver NO will be also be tested through a Cu + dependent GSNO lyase activity.…”