Active site structure in cytochrome c peroxidase and myoglobin mutants: Effects of altered hydrogen bonding to the proximal histidine

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 Angstrom while the Fe-N-epsilon distance in the peroxidases is closer to 1.9 Angstrom. 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 similar to 1.9 to similar to 2.1 Angstrom. Conversely, introduction of a similar strong proximal hydrogen bond in myoglobin shortens Fe-N-epsilon from similar to 2.1 to similar to 1.9 Angstrom. These results correlate well with other biochemical data.