Active Site Structure in Cytochrome c Peroxidase and Myoglobin Mutants:  Effects of Altered Hydrogen Bonding to the Proximal Histidine

Abstract

The globins and peroxidases, while performing completely different chemistry, share features of the iron heme active site: a protoporphyrin IX prosthetic group is linked to the protein by the proximal histidine residue. X-ray absorption spectroscopy provides a method to determine the local structure of iron heme active sites in proteins. Our previous studies using X-ray absorption spectroscopy revealed a significant difference in the Fe-N epsilon bond length between the peroxidases and the globins [for a review, see Powers, L. (1994) Molecular Electronics and Molecular Electronic Devices, Vol. 3, p 211 CRC Press Inc., Boca Raton, FL]. Globins typically have an Fe-N epsilon distance close to 2.1 A while the Fe-N epsilon distance in the peroxidases is closer to 1.9 A. We have proposed [Sinclair, R., Powers, L., Bumpus, J., Albo, A., & Brock, B. (1992) Biochemistry 31, 4892] that strong hydrogen bonding to the proximal histidine is responsible for the shorter bond length in the peroxidases. Here we use site-specific mutagenesis to eliminate the strong proximal hydrogen bonding in cytochrome c peroxidase and to introduce strong proximal hydrogen bonding in myoglobin. Consistent with our hypothesis, elimination of the Asp235-His175 hydrogen bond in CcP results in elongation of Fe-N epsilon from approximately 1.9 to approximately 2.1 A. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N epsilon from approximately 2.1 to approximately 1.9 A. These results correlate well with other biochemical data.

Active Site of Cytochrome cbb3

“…However, a similarity in this respect between cytochrome cbb 3 and cytochrome c peroxidase (CcP) is of interest. The catalytic triad in CcP involves a hydrogen bond from the proximal histidine ligand of the protoheme to an aspartate residue (42), and a direct effect of such hydrogen bonding upon the length of the Fe-His bond has been studied in detail (43). EPR experiments of CcP also yielded rhombic heme signals, and mutations of the aspartate led to results very similar to those presented here (42).…”

Muskox myoglobin: purification, characterization and kinetics studies compared with cattle and water buffalo myoglobins

“…Mb exhibits pseudoperoxidase activity in the presence of H 2 O 2 , catalysing the oxidation of various compounds. Mb pseudoperoxidase activity differs from that of classic peroxidases given the different packing of the haem group within the different protein globules . In particular, myoglobin has low pseudoperoxidase activity, although it is still somewhat considerable because of its higher concentration in skeletal muscles .…”

“…Mb pseudoperoxidase activity differs from that of classic peroxidases given the different packing of the haem group within the different protein globules. 27 In particular, myoglobin has low pseudoperoxidase activity, although it is still somewhat considerable because of its higher concentration in skeletal muscles. 9 This activity is of interest in meat conservation because it could influence oxidation in meat systems.…”

Unusual proximal heme pocket geometry in the deoxygenated Thermobifida fusca: A combined spectroscopic investigation

“…Several studies in past years have revealed consistent differences between the structure of the heme in globins and peroxidases [31]. As shown from XAS and high resolution X-ray structures, globins typically have an Fe−N h distance of about 2.1 Å while in the peroxidases this distance is typically shorter by about 0.2 Å, resulting in a typical Fe−N h distance of about 1.9 Å [31][32][33][34][35][36][37][38] (see also Table 3). This was proposed to be crucial to explain the different reactivity observed between the two groups of proteins [34,35].…”

“…As shown from XAS and high resolution X-ray structures, globins typically have an Fe−N h distance of about 2.1 Å while in the peroxidases this distance is typically shorter by about 0.2 Å, resulting in a typical Fe−N h distance of about 1.9 Å [31][32][33][34][35][36][37][38] (see also Table 3). This was proposed to be crucial to explain the different reactivity observed between the two groups of proteins [34,35]. A significant feature of the peroxidase structures (either Cytochrome C peroxidase, Ccp, or Horseradich peroxidase, Hrp) is the presence of strongly hydrogen-bonding proximal histidine [38].…”