R. Daniel Libby

Research

Mechanistic Studies of Chloroperoxidase (CPO)


Background: Chloroperoxidase (CPO) occupies a key position among hemoproteins. CPO catalyzes reactions characteristic of peroxidases, hydrogen peroxide supported oxidation (peroxidatic reactions, Eqn. 1) and iodination (halogenation reactions, Eqn. 2, X- = I-) of a variety of organic substrates (peroxidatic substrates, PSH2, and halogenation substrates, RH2).

PSH2 + H2O2 PS + 2 H2O                    (1)
H2O2 + X- + RH2 + H+RHX + 2 H2O    (2)
however, unlike most other peroxidases, CPO also catalyzes the dismutation of hydrogen peroxide (catalatic reaction, Eqn. 3), chlorination and bromination reactions (Eqn. 2 X- = Cl- and Br-), and some cytochrome P450 mono-oxygenase type reactions (Eqn. 4, R = oxygen acceptor substrate).
2 H2O2 O2 + 2 H2O                              (3)

H2O2 + R + H+ RO + H2O                    (4)

Thus, it is likely that a thorough understanding of the structure and mechanism of action of CPO would provide valuable insight into the behavior and interrelationships of catalases, cytochrome P 450 mono-oxygenases and peroxidases other than CPO.

A general outline of the pathways of reactions catalyzed by CPO is given in Eqns. 5 -> 14.

E + H2O2 Cpd I + H2O                          (5)

Cpd I + PSH2E + PS + H2O                   (6)

Cpd I + H2O2E + O2 + H2O                   (7)

Cpd I + R E + RO                                  (8)

Cpd I + X- EOX                                     (9)

EOX + PSH2E + PS + X- + H2O           (10)

EOX + RH2 + H+E + RHX + H2O        (11)

EOX + H2O2E + O2 + X- + H2O          (12)

EOX + R E + RO + X-                          (13)

EOX + X-+ 2 H+E + X2 + H2O             (14)

In these equations, E represents the native enzyme, Cpd I is compound I, an enzymic intermediate that is two electrons more oxidized than E and has incorporated one of the oxygen atoms from the peroxide substrate, EOX is the enzymic electrophilic halogenating intermediate.

Peroxidases and catalases react rapidly with H2O2 and a variety of organic hydroperoxides or peroxyacids to produce Cpd I, Eqn. 5. This is the first step in all CPO reactions. Eqn. 6 completes the classical peroxidatic reaction. Eqn. 7 completes the catalatic reaction. Eqn. 8 completes the peroxygenase reaction and seems to occur by a direct stereospecific oxygen atom transfer mechanism. Finally, Eqn. 9, which initiates the halide dependent reactions, indicates the competition of halide ions with organic substrates and H2O2 for reaction with Cpd I, Eqns. 6-9. Eqn. 10 completes the normal halogenation pathway. Eqns. 11 ,12 and 13 complete the X- dependent peroxidatic, catalatic and peroxygenase reactions respectively. Finally, Eqn. 14 is the pathway for formation of molecular halogen. It should be noted that various alkyl hydroperoxides (ROOH’s) and peroxy acids can replace H2O2 in reactions Eqns. 5, 7 and 12.

Goals: The primary goal of this project is to obtain detailed knowledge of the specifics of the many reactions catalyzed by CPO and how the various pathways relate to each other. We are employing the wide range of catalytic activities of CPO to advantage by using one activity to probe another.

Progress: Thus far, we have succeeded in defining the mechanistic interrelationship between chloride dependent and independent CPO pathways and in defining the conditions that favor formation of an enzyme generated Cl2 intermediate. Our results have produced the first documented example of competitive activation of an enzyme.1 We have also presented the first evidence for a peroxidase reaction in which the initial reaction of the enzyme with hydrogen peroxide is not clearly rate limiting.2 In addition we have reported the first evidence for kinetically significant involvement of a freely dissociable, enzyme generated, oxidized halogen species, Cl2, in a peroxidatic reaction3 and demonstrated that free Cl2 is not a significant intermediate in CPO catalyzed reactions of usual good halogenation substrates.4The latter results have settled a long standing controversy concerning the potential involvement of free halogen species in CPO catalyzed chlorination reactions. Most recently, we have defined the competitive activation of CPO peroxidatic reactions by Br-.5 The Br- activation mechanism is similar to that for Cl- activation, but the kinetics are considerably more complex and include significant involvement of Br2 as a reaction intermediate at low substrate concentrations .

Current Work: Having clarified the halide involvement in CPO peroxidatic and halogenation reactions, we are now focusing on the halide independent peroxidatic reaction pathway. (Eqns. 5 & 6) We are defining the peroxidatic reaction conditions and substrate structural characteristics that favor one electron reaction steps, which produce free radical intermediates, over their two electron alternative processes, where no free radicals are produced.

From mechanism-based suicide inhibitor studies with horseradish peroxidase (HRP), Ortiz de Montellano et. al. have proposed a model for heme enzyme reactivity .6,7,8 In their model, one electron reactions catalyzed by peroxidases take place at the d-edge of the heme while oxygen atom transfers and other two electron processes occur at the iron center of the heme group. They suggested that with HRP the protein structure effectively blocks substrate access to the heme-iron thus HRP can catalyze only one electron processes. Also, since the d-edge of the hemes of several cytochrome P-450 enzymes are not accessible to the substrate, these enzymes cannot catalyze one electron processes. Thus, the heme-iron vs. heme-edge model explains differences in reactivity among HRP, CPO, catalase and cytochrome P-450systems largely on he basis of variations in heme accessibility.6,9,10. The versatility of CPO as a catalyst was proposed to arise from the accessibility to substrates of both its heme d-edge and heme-iron. If this proposal is correct, the key question is, which factors favor heme-edge reactions and which favor heme-iron reactions?

However, more recent inhibitor studies with CPO indicate that the heme-iron is generally accessible to substrates, but that the d-edge of the heme was modified only in reactions involving the relatively unhindered azide ion.9 A recent crystal structure also indicates that the heme edge is not generally available to solvent in the native form of the enzyme.11 Finally, Casella et. al. have used CPO phenol peroxidation as a tool to investigate the substrate structural characteristics that favor binding to the enzyme.12 Their results suggest that both steric and electrostatic factors have significant effects on binding. They report that phenols with positively charged p-substituents or with branched chain p-substituents are not substrates for CPO. These compounds are substrates for HRP. They also saw stereoselectivity with some chiral substrates. They found that L-N-acetyltyrosine is a substrate while the D- enantiomer is not.

We expect that the extent of radical formation should be affected by the ability of the molecular structure of the substrate to accommodate a free radical. To these ends, CPO catalyzed reactions of a large number of peroxidatic substrates are being investigated and each individual process is being probed with trapping agents for non-enzymic (enzyme generated, but not enzyme bound) free radical intermediates. One electron and two electron oxidation potentials of each substrate are being determined under the conditions used for the enzymatic reactions. Although thermodynamic behavior does not necessarily correlate with reaction rates, when the factors that affect thermodynamic stabilities of reactants and products also affect a reaction’s transition state, kinetics and thermodynamics can parallel each other. There is some evidence for the correlation of redox potentials of CPO substrates with their kinetic behavior. In halogenation reactions catalyzed by CPO, the relative rates of halogenation correlate with the ease of oxidation of the halide ion. Maximum velocities for these reactions follow the order I- > Br-> Cl-.2,13 Dependence of the rates of reaction of phenols and arylamines in CPO catalyzed reactions on the ease of removal of the first electron from the substrates have been reported.12,14,15 Consequently, the extent of radical formation for a substrate in a peroxidatic reaction catalyzed by CPO could be at least partially related to the difference in the potential for the removal of the first electron from the substrate and that for removal of the second electron. This effect could arise if the heme-iron reaction partitioned between one electron or two electron processes depending on substrate reactivity. A direct correlation between the extent of radical formation with the difference between the one and two electron oxidation potentials of the substrates would be difficult to rationalize with the heme-iron heme-edge model for heme enzyme reactions. However, a lack of correlation would support that model.

In the course of this study, a number of compounds are being characterized as to the differences in their reactivity with small molecules vs. enzymes. The steric and electrostatic limitations on the reactivity of these compounds as well as the specific products formed from their reactions with various oxidizing agents are being characterized. Thus, a secondary result of this work is the development of generally useful probes for detecting the kinetically significant involvement of non-enzymic free radical species in any enzyme catalyzed reaction whose rate can be easily measured.

We have also begun HPLC product distribution studies that compare the product mixtures from known radical reactions catalyzed by HRP with CPO catalyzed reactions of the same substrates. Preliminary results suggest that this technique may be useful in determining the extent of radical involvement with substrates that are not amenable to trapping studies.

Priorities and Future Research Plans: As indicated above we have begun work on halide independent peroxidatic CPO pathways, but there is much left to do. The focus of this study is to determine the extent of the involvement of substrate based free radical intermediates in halide independent peroxidatic reactions catalyzed by CPO. If CPO compound I has the capability of accepting either one or two electrons at a time, the extent of involvement of the one electron pathway should be affected by the substrate's ability to accommodate the intermediate radical. Results from these studies should help us judge the potential range of reactivities of CPO compound I. Preliminary results suggest that substrate structure does affect the extent of formation of free radical intermediates, but more data are needed to characterize the effects fully.

Other future studies will focus on elucidating the effects of halide ions on the CPO catalatic reaction.

Student Involvement: Over the last 20 years at Colby, Barnard and Moravian this project has involved 35 undergraduate students. (see list below) Undergraduate students have collected all of the data included in the papers on this project. In general the techniques required for this project are within the capabilities of a student who has completed introductory organic chemistry. In fact most of my research students have worked in the summer after their sophomore year or in the beginning of their junior year. The background in enzyme kinetics is covered in individual tutorials or small group discussions. I spend a short period of time training each student, and then together we decide on the particular aspect of the project that most interests the student. We then work together in planning experiments and discussing data analysis. Thus, my undergraduate research group is organized very much like a graduate research group.

Funding Support: This project was supported by an NSF-RUI grant through April 2001.
 
Moravian 

1992 - Present 

Colby 

1985 - 1992 

Barnard 

1982 - 1985 

Shirin Arastu 

Darrell Campanella 

Jeremie Eckhaus 

Tiffany Fries 

Megan Hahn 

Stephanie Horne 

Eman Jarrah 

Veronica Jaramillo 

Julie Jones 

Erica Landes 

Dan Moneo 

Krisa Murray 

Brian Rauch 

Michael Reyda 

Susan Schaivo 

Kris Silver 

Jason Switzer 

Lisa Tarricone 

Leah Williams 

George Young 

Tina Beachy 

Bob Cobuzzi 

Toby Emerson 

Mike Genco 

Sue Gerstberger 

Norm Navarro 

Kathy Phipps 

Dave Provencial 

Nicola Rotberg 

Heidi Senkler 

Amy Shedd 

Andri Smith 

Sharon Friedman 

Robyn Goldowski 

Jasmin Kakoo 

Yin Yin Shang 

Nancy Sun 

Theresa White 

Gabrielle Yen 

References:

  1.  Libby, R.D., *Rotberg, N.S., *Emerson, J.T., *White, T.C., *Yen, G.M.,  *Friedman, S.H., *Sun, N.S. and *Goldowski, R., (1989)  J. Biol. Chem., 264, 15284-15292.
  2.  Libby, R.D. and *Rotberg, N.S.  (1990) J. Biol. Chem. 265, 14808-811.
  3.   Libby, R.D., *Shedd, A.L., *Phipps, A.K., *Beachy, T.M. and *Gerstberger, S.M. (1992) J. Biol. Chem.  267, 1769-75.
  4.  *Beachy, T.M., *Phipps, A.K. and Libby, R. D.  (1996) J. Biol. Chem. 271, 21820-7.
  5.  R. Daniel Libby, Jeremiah Eckhaus* and Daniel A. Moniot*,  (1999) manuscript in preparation for submission to The Journal of Biological Chemistry.
  6.  Ortiz de Montellano, P. R., Choe, Y. S., DePhillis, G. and Catalano, C. E.,  (1987) J. Biol. Chem.  262,  11641-11646.
  7.  Ator, M. A. and Ortiz de Montellano, P. R.  (1987) J. Biol. Chem.  262,  1542-1551.
  8.  Harris, R. Z., Newmeyer, S. L. and Ortiz de Montellano, P. R., (1993) J. Biol. Chem.  268,  1637-1645.
  9.  Samokyszyn, V. M. and Ortiz de Montellano, P. R. (1991) Biochemistry  30, 11646-11653.
  10.  Ortiz de Montellano, P. R.  (1992) Ann. Rev. Pharm. Tox..  32,  89-107.
  11. Sundaramoorthy M, Terner J, Poulos T. L. (1995) Structure 3, 1367-1377.
  12. Casella, Luigi; Gullotti, M., Selvaggini, C., Poli, S., Beringhelli, T. and Marchesini, A.  (1994) Biochemistry  33, 6377-86.
  13.  Libby, R.D., Thomas, J. A., Kaiser, L. W. and Hager,  L. P. (1980) J. Biol. Chem.  257,  5030-5037.
  14.  Casella, L., Gullotti, M, Ghezzi, R., Poli, S., Beringhelli, T., Colonna, S. and Carrea, G.  (1992) Biochemistry  31, 9451-9.
  15.  Colonna, S., Gaggero, N., Manfredi, A., Casella, L., Gullotti, M., Carrea, G. and Pasta, P. (1990) Biochemistry  29, 10465-8.