CYTOCHROME C PEROXIDASE
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Cytochrome c peroxidase (CcP) is one member of a huge family of heme peroxidases. These enzymes link reduction of hydroperoxides (i.e. ROOH to ROH + HOH) to oxidation of an exogenous electron donor. The reaction with peroxide is quite similar for all of the heme peroxidases, and the
heme binding pocket is highly conserved among heme peroxidases. The electron donor is characteristic for each peroxidase, and considerable variability is seen in the nature of the donor. This variability reflects the diverse ways that organisms link the oxidizing power of peroxide to various metabolic tasks.

CcP does not have a well defined metabolic role, but seems to be designed to detoxify hydrogen peroxide formed in the intermembrane space of mitochrondria during aerobic metabolism. It does so using ferro-cytochrome c in the intermembrane space as the electron donor. Thus, CcP holds little interest from a metabolic perspective, but its ability to promote rapid electron transfer from cytochrome c makes it a useful structural model for the more complex electron transfer systems that participate in aerobic metabolism. Moreover, its reaction with peroxide is quite similar to other known peroxidases, and it is widely viewed as an archetype for this reaction.

The catalytic cycle of heme peroxidases begins with the coordination of peroxide to the ferric heme. This reaction is near (but below) the diffusion limit. Bound peroxide undergoes rapid heterolytic cleavage, producing a molecule of water and the semi-stable intermediate referred to (for historical reasons) as Compound I. The reaction involves transfer of a proton from peroxide O1 to O2, followed by O-O bond breaking. Departure of O2 as water leaves O1 coordinated to the heme with only six electrons. It completes its octet by abstracting the two most readily available electrons from the enzyme. One electron is removed from the iron, creating an oxy-ferryl (Fe=O) center. For most peroxidases, the second electron is removed from the porphyrin ring, creating a porphyrin pi-cation radical. For CcP, however, the second electron is removed from a Trp residue (Trp 191) that is in van der Waals contact with the heme. Oxidation of Trp 191 produces an indolyl cation radical that is stable in solution for several hours at room temperature. Click here for a picture of how the reaction sequence might look.

The second stage of the peroxidase catalytic cycle involves reduction of Compound I by an electron donor. For CcP, this occurs by the sequential reaction of Compound I with two molecules of ferro-cytochrome c. Reaction with the first molecule of cytochrome c produces Compound II and a molecule of ferri-cytochrome c. Subsequent reaction of Compound II with ferro-cytochrome c returns CcP to the resting ferric state, producing a second molecule of ferri-cytochrome c and releasing O1 of peroxide as water. Both reactions involve reversible formation of a CcP:Cc complex, followed by electron transfer.


STRUCTURE:FUNCTION RELATIONSHIPS FOR CCP



Several aspects of the mechanism of CcP are extremely interesting from the standpoint of structure:function relationships. I have chosen to focus on three specific areas, and my research has benefitted from collaborations with many talented individual in several other groups.

Perhaps the most provocative issue to address is how electrons can move rapidly (approximately 10^6 s-1) from ferro-cytochrome c to the oxidized form of CcP. It is crucial to understand how both the rate and specificity of the reaction are attained.

A second important issue is how the enzyme manages to create an indolyl radical in solution that has has a half life of more than 4 hr at room temperature. This alone seems remarkable, but it is even more remarkable when one considers the fact that this stable radical exists in van der Waals contact with a porphyrin ring, and in close proximity to several Tyr residues, which would be rapidly oxidized by a normal Trp radical. That is, how does the local enzyme structure stabilize the Trp 191 radical?

Another important issue is how the ferric protoporphyrin IX prosthetic group of heme peroxidases reacts so rapidly with hydroperoxides, while in the globins, the same peroxidase is approximately 5-orders of magnitude slower. That is, what factors determine the influence of local protein environment upon heme reactivity?

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