Design and Biosynthesis of Heterodinuclear Metal Centers
Figure 1: Biosythetic Models of CcO in Mb
Figure 2: Biosynthetic Models of NOR in Mb
Figure 3: Heme-Mn(II) Center of CcP Designed to Mimic MnP
Most metalloenzymes studied to date contain homonuclear metal sites, such as the CuA and Fe-S clusters. However, many new examples of heteronuclear-containing proteins, such as the heme-copper center in cytochrome c oxidase (CcO), the heme-non-heme iron center in nitric oxide reductase (NOR) and the heme-manganese center in manganese peroxidase (MnP), have been discovered. Most of these metalloproteins are not well understood due to the inherent complexity that results from multiple and different metal ions. Therefore, understanding this class of metalloproteins represents one of the greatest challenges facing bioinorganic chemists. To meet the challenge, we have:
  1. Designed and synthesized a heme-copper center in myoglobin (1) and cytochrome c peroxidase (2) that mimics the center found in CcO (See Figure 1). Because CcOs are large membrane bound proteins, identification of structural features responsible for their enzymatic activities has been difficult, and their reaction mechanisms remain controversial. Being much smaller, more stable, and free from other chromophores, our biosynthetic models allowed us to:
    1. clearly define the roles of the CuB center (2, 3), chloride (4), and heme type (5) in the function of CcO.
    2. demonstrate the importance of secondary coordination, specifically hydrogen bonding networks, in fine-tuning the functional properties of CcO (6).
    3. show that tyrosine and its positioning in the active site is critical for complete oxidase activity (7, 8), and that an unnatural His-Tyr mimic enhances the activity (9).
    4. establish the role of conserved tyrosine as an H-radical donor by directly observing the tyrosyl radical under physiological oxygen reduction conditions (10)
    5. established the role of redox potential of the heme group in the O2 reduction activity (11)


  2. Designed and synthesized a heme-non-heme center in myoglobin to mimic that in NOR, using energy minimized computer modeling and site directed mutagenesis, respectively (Figure 2). Our engineered myoglobin is both a structural and functional mimic of NOR (8). Furthermore, by engineering the interface between our model and its physiological redox partner, cyt b5, we have designed a system with O2 reduction rates reaching that of native systems (12). The biosynthetic models have allowed us to:
    1. clearly define the roles of FeB center and coordinating glutamate towards the function of NORs (8).
    2. demonstrate the importance of secondary coordination, specifically hydrogen bonding network, towards NOR activity by introducing an additional glutamate in the active site (13).
    3. model a recently discovered NOR (gNOR), with varied combination of metal binding residues (14).
    4. report an unprecedented Fe-NO stretching frequency using our model systems, which has helped to shed light on the mechanism of NO reduction (15).
    5. reveal the electronic structural and functional properties of the FeB-nitrosyl complex by replacing Fe based-heme with isostructural Zn protoporphyrin IX (16).
    6. show that the trans mechanism is favored for NO reduction in NORs (17).
    7. use most recent spectroscopic techniques, such as NRVS to obtain information on the modes of NO binding to the non-heme iron center, which has been difficult to obtain, due to the presence of heme center that binds NO strongly (18).


    The proposed active site of NOR is similar in structure to the one found in CcO, as the copper center in CcO is replaced by a non-heme iron center in NOR. Therefore, we are focusing on a fundamental question of why the protein, when using copper (as in CcO), is proficient at O-O bond cleavage and when using iron (as in NOR), is efficient at N-N bond formation. Towards this, we have shown that the heme-copper model we have made in section 1 above, displays NOR activity (19), similar to some native CcOs.


  3. Designed and synthesized a heme-Mn(II) center in cytochrome c Peroxidase(CcP) (20) which serves as a structural and functional mimic of manganese peroxidase (MnP) (figure 3), a heme peroxidase involved in biodegradation of lignin and bioremediation of aromatic pollutants. We take advantage of the simple and efficient recombinant protein production system that is available for CcP, as well as the striking structural similarity between CcP and MnP. The first design was then improved through rational design to gain 2.5-fold higher activity, making this the best protein model of MnP reported so far (21). The knowledge gained from this study can be utilized to design new, inexpensive heme peroxidases that are more effective against tougher organic wastes such as polychlorinated biphenyls. From the process, we elucidate the role of tryptophans (22) and tyrosines (23) in peroxidase activity, as well as hydrogen bonding to reactive intermediates (24).
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