Heme orientation

J. Thomas and G. N. LaMar, Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton NMR spectroscopy, Biochem, Biophys. Res. Commun. 119, 640-654 (1984). 

Another significant difference between the large- and small-subunit enzymes lies in the fact that the heme d of HPII and PVC is flipped 180° relative to the heme b moiety of BLC, MLC, SCC-A, and PMC (Fig. 13). This is clearly a function of the residues that form the heme pocket, although attempts to force a change in heme orientation in HPII by mutating residues that interact with the heme were imsuccessful. The heme is situated in the (3-barrel and has interactions with the wrapping domain and with the amino-terminal arm of the R-related subunit. The dimensions of the pocket demand that heme bind in its final conformation and that flipping once inside the pocket not be possible. 

Fig. 12. A portion of the 500 MHz DEFT-NOESY spectrum of NPl-ImH, recorded at 30°C in 50 mM phosphate buffer, pH 7.0, D2O. Note the strong EXSY cross peak between the 3-Me signal at 24.9 ppm and a minor 3-Me signal at 26.4 ppm. This is not the other heme orientational isomer, as is evident by comparison of spectrum (a) of Fig. 11. This chemical exchange is also observed for many protein side chains, suggesting fluxionality of some loop, probably the A-B loop that passes close to the heme. In the text, the small peak is defined as species Y, and the large as species X. ...

These results with regard to heme disorder differ from those observed in solution by NMR spectroscopy, where one heme orientation is at least highly favored (Section 11,E,2). The difference in conditions (high salt, PEG) may be involved in the fact that different ratios of the two heme orientations are observed in the crystalline state than in solntion. The difference in heme orientation ratios of 1.5 1 and 10 1, for example, represents a AG difference of only 4.9 kJ/mol. 

Makinen, M. W., Schichman, S. A., Hill, S. C., and Gray, H. B. (1983). Heme-heme orientation and electron transfer kinetic behavior of multisite oxidation-reduction enzymes. Science 222, 929-931. 

In the meantime, Fritsch, Buchanan and Michel examined heme orientation in the crystals of Rp. viridis reaction centers. Absorption spectra of the crystals poised at different redox potentials were obtained using plane-polarized light. The midpoint potentials were quantitatively determined by fitting the absorbances at 552, 553, 556 and 559 nm to Nemst functions with two exponential terms. Based on the known orientations of the hemes in the reaction-center crystal, the calculaed potentials were found to correspond to the specific hemes in the following sequence ... 

Fig. 11. Model of the tetraheme arrangement in the cytochrome subunit in Rp. viridis consistent with available evidence based on spectral, electrochemical and onentational properties of the hemes. Heme orientations adapted from Alegria and Dutton (1991) II. Langmuir-Blodgeti monolayer films of the Rhodopseudomonas viridis reaction center determination of the order of the hemes in the cytochrome c subunit. Biochim Biophys Acta 1057 271.

NMR study of Mb revealed that there are two interconverting protein forms in solution at equilibrium, which differ in the orientation of the heme by a 180° rotation about the 5,15-meso axis (Fig. 19). - The dominant form possesses the same heme orientation as found in the single crystal (A of Fig. 19) and is called the major form. The protein with the reversed heme orientation (B of Fig. 19) is called the minor form. Although the concentration of the minor form relative to the major form is small in native Mb, it can be raised using the reconstitution technique. The reconstitution of apoprotein with heme initially... 

The reaction of apoMb with hemin yields 1 1 mixture of the two isomers possessing the two heme orientations differing by a 180° rotation about the S, l5-meso axis (Fig. 19) 85.183-186 -phese isomers can be most conveniently identified by H NMR spectra of metMb(CN—) i83, i84 paramagnetically... 

Myoglobin reactivity with H202 is very slow compared to peroxidases. The origin of this poor reactivity was thought to be due to the distal heme orientation... 

CD can be used to monitor the phenomenon of heme isomerism in heme proteins. " The isomerism, discovered by LaMar and co-workers, results from the presence of two forms of myoglobin which differ by 180° in the heme orientation about the a, y-methine carbon axis. The two isomers are present in a 9 1 ratio at equilibrium in carbonmonoxymyo-globin (MbCO) but are in a 1 1 ratio in a sample freshly reconstituted from apomyoglobin and heme. Freshly reconstituted MbCO has a Soret CD band with only about half the amplitude of the native form, " suggesting that the minor isomer (at equilibrium) has only a weak Soret CD spectrum. The major form has a strong positive Soret CD band, whereas the minor form has a weakly negative Soret CD band. These results and the CD of the heme undecapeptide from cytochrome indicate that the origin of the Soret CD band must be more complex than proposed by Hsu and Woody, ... 

ESR anisotropy and linear dichroism of Mb-DDAB films showed that the iron heme gronp was oriented in snrfactant films, but with relatively broad angular distributions [20]. Mb heme orientation angles were close to 60° irrespective... 

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