Hybrid Heme Proteins

      for Redox Catalysis

by synthesis, reconstitution, mutation

 

Photoactive heme proteins.  

       Harry Gray pioneered the use of photo-active Ru(diimine) complexes to initiate and measure the rates of electron transfers in metalloproteins, and it has become an important tool in bioinorganic chemistry. Such use is based on the redox activity of a long-lived (ca. 700 ns) triplet excited state formed by photo-excitation into a  metal-to-ligand charge transfer band.  The triplet state can act as both a reductant (E = -0.77 V) or an oxidant (E = +.86 V); flash/quench techniques allow the more powerful redox species Ru3+ (E = +1.26 V) or Ru1+ (E = -1.35 V) to be generated.

       We use these methods to initiate and follow redox-induced transformations important to heme-based catalysis. Our approach is to hardwire the Ru complex directly to the heme active site -- as in the heme protein myoglobin shown below.

      First, we synthesized a pendant-arm heme cofactor that can be reconstituted with apomyoglobin, reforming the native myoglobin structure but with a photoactive Ru complex covalently linked --or hardwired-- to the heme active site.

 

Reaction scheme for making photoactive heme cofactors

adapted from Hamachi, Tanaka, Shinkai Chem Letts. 1993

     When the photo-active heme is reconstituted into apomyoglobin, a hybrid RuC7Mb is obtained, with an absorbance spectra virtually identical to the sum of normal Mb and Ru(bpy)32+.  With this hybrid, photo-initiation can generate FeIV=O  formation and look at the ability of the ferryl and porphyrin radical cation to oxidize nearby protein residues.

        The oxygen-binding proteins myoglobin and hemoglobin short-circuit any possible oxidative chemistry of high-valent Fe hemes by preferentially oxidizing protein residues instead. In a recent paper (Immoos, Inorg. Chem. 2004), we report the first determination of the rates of such a short-circuit reaction in myoglobin . Oxidative flash/quench experiments using RuC7Mb generate a porphyrin cation radical (P•+), which in a subsequent step, can oxidize either the Fe3+ (to give FeIV=O) or a protein residue. As determined by comparative rate and product formation, protein oxidation dominates over that of Fe. Strong EPR signals attributable to tyrosine and tryptophan radicals were observed after flash-quench/freezing. A similar protein-shortcircuit oxidation to a catalytically active Trp is crucial to the activity of cytochrome c peroxide, CcP; in ongoing work we have generated and determined the lifetime of the often-invoked porphyrin radical of CcP, never before observed.
 

     Reconstituted oxochlorins.  We use similar methods to make new protein catalysts (artificial enzymes) using heme-altered cofactors . Inspired by the oxygenated heme of the cd1 nitrite reductases (see NiR), we synthesized an oxochlorin-reconstituted cytochrome c peroxidase, MpCcP,  which was the first structurally characterized oxochlorin in a protein (Immoos, JIB. 2002). Although a mixture of R- and S- isomers of the oxochlorin were used, only the S-isomer is found reconstituted in the crystallized protein. The hybrid MpCcP retained 99% wildtype peroxidase activity with cytochrome c.

      We have synthesized several oxochlorin derivatives and reconstituted them into myoglobin and cytochrome c peroxidase.  The reconstituted hybrids are green, and display altered ligand-binding and redox catalytitic properties.  We have crystallographically characterized CcP hybrids using two different regioisomers of mesopone-- in both cases only one enantiomer of the oxochlorin is observed, i.e. the reconstitution itself is enantiomerically selective.

 

 

       Another uncommon aspect of NiR enzymes is the presence of tyrosine residues within the active site. To model this, a distal pocket His52 Tyr mutant of CcP was generated (Bhaskar, JMB. 2003), whose structure revealed a novel covalent bond between the Tyr52 and the indole ring nitrogen of Trp51. The crosslink was shown to result from Fe activation of peroxide, and similar metal-dependent crosslinks have been found in cytochrome c oxidase and tyrosinase metalloproteins. In this case, the cross-link C-N bond itself is unusual, in that the Trp N is highly pyramidalized, with the NE1Trp-CE1Tyr bond bent ca. 600 from the aromatic plane. This, to our knowledge, is the largest deformation of an alkylated indole yet observed.
 

  Selected recent publications on heme protein engineering:

“High Temperature Electrocatalysis Using Thermophilic P450 CYP119: Dehalogenation of CCl4 to CH4” Blair, E.; Greaves, J.; Farmer, P.J. J. Am. Chem. Soc. 2004, 126, 8632-8633.

 “Electron Transfer Chemistry of Ru-linker-(heme)-modified Myoglobin: Rapid Intraprotein Reduction of a Photogenerated Porphyrin Cation Radical” Immoos, C.E.; Di Bilio, A.J.; Cohen, M.S.; Van der Veer, W.; Gray, H.B.; Farmer, P.J. Inorg Chem. 2004, 43, 3593 – 3596.

“A Novel Heme and Peroxide-Dependent Tryptophan-Tyrosine Cross-link in a Mutant of Cytochrome c Peroxidase” Bhaskar, B.; Immoos, C.E.; Shimizu, H.; Sulc, F.; Farmer, P.J.; Poulos, T.L. J. Mol. Biol. 2003, 328, 157-166. 

"Mesopone Cytochrome C Peroxidase: Functional Model of Heme Oxygenated Oxidases" Immoos, C.E.; Bhaskar, B.; Cohen, M.S.; Barrows, T.P.; Farmer, P.J.; Poulos, T.L.  J. Inorg. Biochem. 2002, 91, 635-643.

“Electron Transfer in the Ruthenated Heme Domain of Cytochrome P450BM-3” Sevrioukova, I.F.; Immoos, C.E.; Poulos, T.L.; Farmer, P.J. Isr. J. Chem. 2000, 40, 47-53.