Cytochrome c Peroxidase
Created by Joyce Chung
Cytochrome c peroxidase (1AA4) found in common baker’s yeast, Saccharomyces cerevisiae, is a heme-containing enzyme that catalyzes the oxidation of ferrocytochrome c to ferricytochrome c in the presence of peroxides. In doing so, cytochrome c peroxidase (CcP) detoxifies hydrogen peroxide during aerobic metabolism, releasing water as a product. Cytochrome c peroxidase is normally found in yeast mitochondria. The reactions of CcP occurs in three distinct steps; first forming an intermediate named Compound I (or Complex ES, for enzyme-substrate), then forming Compound II, and finally reducing back to its native state [9]. An unusual feature in peroxidases that is unique to Compound I in CcP is the presence of a long-lived free radical, identified as the side chain of Trp-191. As a 294-residue monomer, CcP is a relatively simple enzyme and thus serves as a useful model to study multi-subunit cytochrome c oxidases that are more complex. Furthermore, the study of cytochrome c peroxidase has contributed to the understanding of how redox partners recognize and bind to each other, and how long distance electron transfer is controlled. Mutant forms of cytochrome c peroxidase (W191G, in which Trp-191 is replaced by Gly-191) have been studied to give insight on creating artificial protein cavities designed to be highly specific for catalysts and receptors. Manipulation of these protein cavities can serve to create therapeutic agents and biosensors.
Cytochrome c peroxidase has a molecular weight of 33418.34 Da and an isoelectric point of 5.18 [2]. The single chain of cytochrome c peroxidase is folded into two distinct domains. It consists of 10 α-helicies that make up about 50% of the molecule and two anti-parallel β sheets and a β sheet that make up about 7% of the molecule. Domain I consists of the N-terminus, helicies A through D, and is situated directly above the heme. Domain II contains helicies F through I, most of the β structure, and is located below the heme. The E helix connects the two domains, and the C-terminus-containing J helix extends from Domain I into Domain II. A large channel (10 Å by 5 Å) forms between the domains. The channel connects the distal side of the heme with the molecule surface and allows for substrates to access the active site. The heme fits between helix B in Domain I and helix F in Domain II [3].
His-175 serves as the heme ligand. His-175 is located on α-helix F and distinguished as the only 310 helix with more than one hydrogen bond. Asn-235 and Trp-191 also play important roles in the enzymatic functions of cytochrome c peroxidase. His-175 is hydrogen bonded to Asn-235, which is hydrogen bonded to Trp-191. These hydrogen bonds reduce flexibility in the His-175, which in turn constrains the high-spin iron in its preferred plane for the formation of Compound I, the enzyme-substrate intermediate formed in the first step of catalysis [3].
During ferrocytochrome c oxidation, the peroxide substrate binds to the distal side of the heme, which is next to a 3-residue turn of helix B containing Arg-48, Trp-51, and His-52. These residues play key roles in the enzymatic functions of cytochrome c peroxidase. His-52 is responsible for deprotonating the peroxide bound to the heme iron, resulting in the formation of the first intermediate of the reaction. Furthermore, by removing a proton from the O2, His-52 creates an anionic ligand that results in the shift of Arg-48 toward the active site. The change in conformation allows Arg-48 to heterolytically cleave the O-O covalent bond [3].
Following the formation of Compound I, the activated complex is reduced to return to its native enzyme structure and ferricytochrome c is oxidized to ferrocytochrome c. Residues with acidic side chains on the surface of cytochrome c peroxidase including Asp-33, Asp-34, Asp-37, and Asp-217 interact with complementary lysine residues on cytochrome c. The alignment of two heme groups coupled with lysine and aspartic acid interactions and hydrogen bonding assist in enhancing the rate of electron transfer [10]. The exact mechanics of this process are still being studied.
As a small monomer, cytochrome c peroxidase functions well as a model enzyme for manipulation. Studies of a single residue substitution mutation (W191G) have provided insight on using modified small enzymes such as cytochrome c peroxidase as therapeutic agents and biosensors through the manipulation of its ligand-binding cavity. In the glycine replaced mutant, residues 190-195 were changed into an open conformation for ligand binding [4]. In the wild-type cytochrome c peroxidase, Trp-191 is hydrogen bound to Asn-235 and plays an important role in the binding of the heme substrate.
Leishmania major peroxidase (3RIV) is a protein similar to cytochrome c peroxidase. Protein BLAST results yield a significantly low value (E = 9.87 * 10-54) and indicate a similar primary structure shared between Leishmania major peroxidase and cytochrome c peroxidase. DALI results show a significantly high value (Z score = 36.6) and indicate similar tertiary structures between the two proteins as well [5,8]. In the parasitic protozoa Leishmania major, Leishmania major peroxidase (LmP) has characteristics of both plant cytosolic ascorbate peroxidase and yeast cytochrome c peroxidase, but primarily serves a biological function similar to that of the latter. Although technically classified as an ascorbate peroxidase, LmP binds with higher affinity to cytochrome c than ascorbate. The affinity for cytochrome c is due to Trp-208, a radical residue essential for the binding of cytochrome c substrate that is conserved from cytochrome c peroxidase in Leishmania major peroxidase. The triple β structure close to the Trp-208 radical in CcP is also conserved in LmP. One β sheet consists of three anti-parallel strands formed from residues 212-214, 222-225, and 229-231, with β turns at residues 216-219 and 225-228. Furthermore, like most heme peroxidases, LmP and CcP share a conserved 10 α-helical structure. Leishmania major peroxidase also displays qualities that more closely mimic ascorbate peroxidase than cytochrome c peroxidase. CcP has a unique insertion between its A and B helicies that provide contact with the cytochrome c substrate. LmP lacks this structure, although its analogous loop is one residue longer than in ascorbate peroxidase [6].