Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase.

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.

Blue ferrocenium azurin: an organometalloprotein with tunable redox properties.

A ferrocene derivative (2-[(methylsulfonyl)thio]ethylferrocene) (1) has been synthesized and incorporated into apo-azurin from Pseudomonas aeruginosa by covalent attachment to the highly conserved Cys112. The resulting artificial organometalloprotein (a protein containing organometallic compounds in the active site) has been characterized by UV-vis, electrospray mass spectrometry, and cyclic voltammetry (CV). Incorporation of 1 into azurin resulted in a higher solubility of the ferrocene group and improved stability of the ferrocenium species in aqueous solution, as shown by a more intense UV-vis absorption and a more reversible CV of the attached ferrocene group, respectively. The incorporation of 1 also increased the reduction potential of the complex from 402 to 579 mV (vs NHE), consistent with the ferrocene group being encapsulated inside the hydrophobic environment of the protein. Modulation of the reduction potential of ferrocene by residues near the secondary coordination sphere has also been demonstrated. Raising the pH from 4 to 9 resulted in a greater than 80 mV decrease in reduction potential of the protein-bound ferrocene (from 579 to 495 mV), while replacing Met121, an amino acid residue in close proximity to the ferrocene group with a positively charged Arg or negatively charged Glu, resulted in the predicted increase or decrease in reduction potential at all pH values. Similarly, substitution of Met121 with a more hydrophobic Leu raised the reduction potential. The increased solubility, stability, and tune-ability of this organometalloprotein make it an ideal choice for carrying out a number of biological reactions, such as long-range electron transfer or sensing. As an example of such applications, stoichiometric oxidation of ferrocytochrome c by the blue ferrocenium azurin was demonstrated.

Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase.

Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).