Preparation and Physicochemical Properties of Supramolecular Hemoprotein Assembly Via Heme-Heme Pocket Interaction

Tuesday, May 13, 2014: 14:40
Bonnet Creek Ballroom X, Lobby Level (Hilton Orlando Bonnet Creek)
T. Hayashi (Osaka University, Department of Applied Chemistry)
Protoheme IX is the simplest and most well-known iron porphyrin in nature and acts as a cofactor in many hemoproteins such as hemoglobin, myoglobin, cytochrome P450, and horseradish peroxidase.  The cofactor, heme b, is bound in the heme pocket via coordination and multiple noncovalent interactions.  Therefore, heme b is released from the heme pocket upon acidic condition to yield the apoprotein.  The apoprotein is capable of binding heme b again at neutral pH value and then the reconstituted protein is available.  To functionalize a hemoprotein, it will be very effective for replacing native heme with an appropriate heme analog.  Our group has recently focused on the following three methods to prepare an artificial prosthetic group.1)   (i) Introduction of functional groups at the terminal of the heme-propionate side chains.  (ii) Modification of the framework of the prosthetic group.  (iii) Replacement of a metal substitution in the core of the porphyrin framework.    

     In our previous work, we prepared several types of supramoelcular hemoprotein assembies via interprotein heme–heme pocket interaction by method (i) vide supra.2)  We first prepared a heme moiety with a maleimide group at the terminal of one of the heme-propionate side chains.  The heme moiety was then introduced to a cysteine residue positioned on the surface of an apocytochrome b562 mutant (H63C) via a covalent linkage.  The heme moiety-linked apoprotein provided a linear hemoprotein assembly determined by an AFM technique.3)  The AFM image clearly demonstrates the 1D protein fiber including more than 100 proteins on a graphite substrate.  Next, we tried to immobilize the hemoprotein polymers onto a gold surface, gold electrode or gold nanoparticle.  Particularly, the heme-attached gold surface provided the hemoprotein assemblies with the number of 6–8 protein layers on the electrode upon the addition of the heme-linked apoproteins.4)  The assembly structure on the electrode was mainly analyzed by the AFM technique and QCM measurement.  Furthermore, the immobilization of zinc cytochrome b562 polymer on a gold electrode gave efficient photocurrent generation in the presence of methyl viologen as an electron mediator.  In addition, it is found that heme-immobilized gold nanoparticles provide a unique self-assembly upon the addition of apohemoprotein dimer.5)  The supramolecular porphyrin array mediated by hemoprotein matrix serves as a new way to create bionanomaterials.  For example, we have prepared a myoglobin–quantum dot conjugate by a heme-immobilized CdTe particle.6)  In this presentation, we report the construction of several supramolecular hemoprotein assemblies in a solution and on a metal surface.     

1)  T. Hayashi, In Handbook of Porphyrin Science; K. M. Kadish, K. M. Smith, R. Guilard, Eds.; World Scientific publishing: Singapore, 2010; Vol. 5, pp 1–69.

2)  K. Oohora, A. Onoda, T. Hayashi, Chem. Commun., 48, 11714–11726 (2012) (Feature Article). 

3)  H. Kitagishi, K. Oohora, H. Yamaguchi, H. Sato, T. Matsuo, A. Harada, T. Hayashi, J. Am. Chem. Soc., 129, 10326–10327 (2007). 

4)  A. Onoda, Y. Kakikura, T. Uematsu, S. Kuwabata, T. Hayashi, Angew. Chem. Int. Ed., 51, 2628–2631 (2012). 

5)  A. Onoda, Y. Ueya, T. Sakamoto, T. Uematsu, T. Hayashi, Chem. Commun., 46, 9107–9109 (2010).

6)  A. Onoda, T. Himiyama, K. Ohkubo, S. Fukuzumi, T. Hayashi, Chem. Commun., 48, 8054–8056 (2012).