Journal of the American Chemical Society
ARTICLE
bis(imidazole)-ligated hemes in the presence of excess imidazole gives
six-coordinate complexes, without displacement of imidazole by the
base.54 Generation of FeIII∼CO2 can be done by working quickly with
static UV, or more conveniently with stopped-flow UVꢀvis, as described
below. Addition of up to 1 equiv of excess base caused no further
UVꢀvis spectral changes. The addition of 1 equiv of triflic acid to
solutions of FeIII∼CO2 cleanly regenerates FeIII∼CO2Hþ. The spectra
show no dependence on [MeIm] up to 20 mM (the highest concentra-
tion tested). UVꢀvis (MeCN), Q-bands 535 (7700), 616 (3800).
5.3. Stopped-Flow Kinetics. All kinetics experiments were
carried out as previously described.15,24,25 Full details are given in the
Supporting Information.
transfer of eꢀ and Hþ in the same kinetic step, while PCET refers to any
process affected by transfer of both electron(s) and proton(s).
(6) (a) Bertero, M. G.; Rothery, R. A.; Boroumand, N.; Palak, M.;
Blasco, F.; Ginet, N.; Weiner, J. H.; Strynadka, N. C. J. J. Biol. Chem.
2005, 280, 14836–14843. (b) Crane, B. R.; Arvai, A. S.; Ghosh, D. K.;
Wu, C.; Getzoff, E. D.; Stuehr, D. J.; Tainer, J. A. Science 1998,
::
279, 2121–2126. (c) Brzezinski, P.; Adelroth, P. Curr. Opin. Struct. Biol.
2006, 16, 465–472.
(7) (a) Haas, A. H.; Sauer, U. S.; Gross, R.; Simon, J.; M€antele, W.;
Lancaster, C. R. D. Biochemistry 2005, 44, 13949–13961. (b) Lancaster,
C. R. D.; Sauer, U. S.; Groβ, R.; Haas, A. H.; Graf, J.; Schwalbe, H.;
M€antele, W.; Simon, J.; Madej, M. G. Proc. Natl. Acad. Sci. U.S.A. 2005,
102, 18860–18865.
(8) (a) Sharp, K. H; Mewies, M.; Moody, P. C. E.; Raven, E. L. Nat.
Struct. Biol. 2003, 10, 303–307. (b) Macdonald, I. K.; Badyal, S. K.;
Ghamsari, L.; Moody, P. C. E.; Raven, E. L. Biochemistry 2006, 45,
7808–7817.
5.4. Kinetic and Equilibrium Measurements by Stopped-
Flow. The determination of the pKa of FeIII∼CO2Hþ, confirmation of
ꢀ
the redox FeIII/II∼CO2H and FeIII/II∼CO2 redox potentials by
equilibration with Cp*2Fe, and all of the kinetic studies were done using
an OLIS RSM-1000 stopped-flow rapid-scanning spectrophotometer. In
a typical procedure, a solution of FeIII∼CO2, 0.1 nBu4NPF6, and 5 mM
MeIm in MeCN was freshly generated from FeIII∼CO2Hþ and 1 equiv
of DBU and then was loaded into one syringe. The other syringe was
loaded with a similar solution of 10ꢀ45 equiv of iAscHꢀ in MeCN/0.1
nBu4NPF6/5 mM MeIm. The kinetic data were analyzed using a first-
order model. Experimental details and plots are given in the Supporting
Information.
(9) See, for instance: Guallar, V. J. Phys. Chem. B 2008,
112, 13460–13464.
(10) (a) Kaila, V. R. I.; Verkhovsky, M. I.; Wikstr€om, M. Chem. Rev.
2010, 110, 7062–7081. (b) See also: Chang, H.-Y.; Hemp, J.; Chen, Y.;
Fee, J. A.; Gennis, R. B. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
16169–16173.
(11) (a) Das, D. K.; Medhi, O. K. J. Inorg. Biochem. 1998, 70, 83–90.
(b) Das, D. K.; Medhi, O. K. Indian J. Chem., Sect. A: Inorg., Bio-inorg.,
Phys., Theor. Anal. Chem. 2005, 44A, 2228–2232.
(12) Generated using the PyMOL Molecular Graphics System,
Version 1.2r3pre, Schr€odinger, LLC.
’ ASSOCIATED CONTENT
(13) (a) Rosenthal, J.; Hodgkiss, J. M.; Young, E. R.; Nocera, D. G. J.
Am. Chem. Soc. 2006, 128, 10474–10483. (b) Young, E. R.; Rosenthal, J.;
Hodgkiss, J. M.; Nocera, D. G. J. Am. Chem. Soc. 2009, 131, 7678–7684.
(c) Cukier, R. I.; Nocera, D. G. Annu. Rev. Phys. Chem. 1998, 49, 337–369.
(d) Hodgkiss, J. M.; Rosenthal, J.; Nocera, D. G. In Hydrogen-Transfer
Reactions; Hynes, J. T., Klinman, J. P., Limbach, H.-H., Schowen, R. L.,
Eds.; Wiley-VCH: Weinheim, Germany, 2007; Vol. 2.17, pp 503ꢀ561.
(e) Pressꢀe, S.; Silbey, R. J. Chem. Phys. 2006, 124, 164504–164511.
(14) For related Fe(PPIX) complexes, see: Little, R. G.; Dymock,
K. R.; Ibers, J. A. J. Am. Chem. Soc. 1975, 97, 4532–4539.
(15) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc. 2008,
130, 2774–2776.
Supporting Information. 1H NMR spectrum of FeIII-
S
b
(PPIXMME)(MeIm)2OTf, cyclic voltammograms of iron com-
plexes, pKa and redox titration data, stopped-flow kinetics data,
EPR spectra. This material is available free of charge via the
’ AUTHOR INFORMATION
Corresponding Author
jwarren@caltech.edu; mayer@chem.washington.edu
(16) Asakura, T.; Lamson, D. W. Anal. Biochem. 1973, 53, 448–451.
(17) The large difference between the Q-bands of FeIII∼CO2Hþ
and FeIII∼CO2 is surprising. Similar changes are observed on adding 1
equiv of DBU to the bis(N-MeIm) complex of native PPIX (not
esterified).
(18) Pagola, S.; Stephens, P. W.; Bohle, D. S.; Kosar, A. D.; Madsen,
S. K. Nature 2000, 404, 307–310.
(19) Stynes, D. V.; Fletcher, D.; Chen, X. Inorg. Chem. 1986, 25,
3483–3488.
(20) Reid, L. S.; Taniguchi, V. T.; Gray, H. B.; Mauk, A. G. J. Am.
Chem. Soc. 1982, 104, 7516–7519.
’ ACKNOWLEDGMENT
We gratefully acknowledge financial support from the U.S.
National Institutes of Health (1F32GM095037 to J.J.W. and
GM50422 to J.M.M.) and the University of Washington. We
thank Dr. Stefan Ochsenbein for help obtaining EPR spectra.
’ REFERENCES
(1) (a) Stubbe, J.; Nocera, D. G.; Yee, C S.; Chang, M. C. Y. Chem.
Rev. 2003, 103, 2167–2201. (b) Mayer, J. M. Annu. Rev. Phys. Chem.
2004, 55, 363–390. (c) Huynh, M. H. V.; Meyer, T. J. Chem. Rev. 2007,
107, 5004–5064.
(2) (a) Dunford, H. B. Heme Peroxidases; Wiley-VCH: New York,
1999. (b) Cytochrome P450: Structure, Mechanism and Biochemistry, 3rd
ed.; Ortiz de Montellano, P. R., Ed.; Kluwer Academic/Plenum: New
York, 2005. (c) Rittle, J.; Green, M. T. Science 2010, 330, 933ꢀ937 and
references therein.
(21) CG = 54.9 kcal molꢀ1 in MeCN versus Cp2Feþ/0. Mader, E. A.;
Davidson, E. R.; Mayer, J. M. J. Am. Chem. Soc. 2007, 129, 5153–5166.
(22) (a) De Santis, G.; Fabbrizzi, L.; Licchelli, M.; Pallaviicini, P.
Inorg. Chim. Acta 1994, 225, 239–244. (b) Herres-Pawlis, S.; Verma, P.;
Haase, R.; Kang, P.; Lyons, C. T.; Wasinger, E. C.; Fl€orke, U.; Henkel,
G.; Stack, T. D. P. J. Am. Chem. Soc. 2009, 131, 1151–1169.
(23) (a) Waidmann, C. R. PhD. Thesis, University of Washington,
Seattle, WA, October 2009. (b) Reference 3, Section 5.9.
(24) (a) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc. 2008,
130, 7546–7547. (b) Warren, J. J.; Mayer, J. M. J. Am. Chem. Soc.
2010, 132, 7784–7793.
(25) (a) Manner, V. W.; Dipasquale, A. G.; Mayer, J. M. J. Am. Chem.
Soc. 2008, 130, 7210–7211. (b) Manner, V. W.; Mayer, J. M. J. Am.
Chem. Soc. 2009, 131, 9874–9875.
(26) Karl Fischer titration shows that the minimum water content is
6 ꢁ 10ꢀ4 M, roughly the same concentration as that of iAscDꢀ used in
this study.
(3) Warren, J. J.; Tronic, T. A.; Mayer, J. M. Chem. Rev. 2010, 110,
6961–7001.
(4) (a) Nakanishi, N.; Rahman, M. M.; Sakamoto, Y.; Miura, M.;
Takeuchi, F.; Park, S.-Y.; Tsubaki, M. J. Biochem. 2009, 146, 857–866.
(b) Njus, D.; Wigle, M.; Kelley, P. M.; Kipp, B. H.; Schlegel, H. B.
Biochemistry 2001, 40, 11905–11911. (c) Nakanishi, N.; Takeuchi, F.;
Tsubaki, M. J. Biochem. 2007, 142, 553–560.
(5) CPET (coined in: Costentin, C.; Evans, D. H.; Robert, M.;
Savꢀeant, J.-M. J. Am. Chem. Soc. 2005, 127, 12490–12491) implies
8550
dx.doi.org/10.1021/ja201663p |J. Am. Chem. Soc. 2011, 133, 8544–8551