Incorporating anomalous scattering centres into macromolecules.

The widespread application of multiwavelength anomalous diffraction (MAD) for phase evaluation has been hampered in the past by the small selection of anomalous scattering centres that could be introduced into macromolecules. Recently, the use of chemical modification, protein engineering or biosynthetic labelling has provided suitable tools to overcome the previous limitations, thereby making most structural analyses amenable to a MAD approach.

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

Site-specific cross-linking as a method for studying intramolecular electron transfer.

Site-directed mutagenesis has been used to introduce cysteine residues into yeast cytochrome c peroxidase and yeast cytochrome c for the purpose of forming site-specific cross-linked intermolecular complexes. This enables the formation of well-defined homogeneous covalently linked complexes for the purpose of relating structure to intramolecular electron transfer. Two complexes have been prepared and analyzed. Complex I has an engineered cysteine at position 290 near the C-terminus of the peroxidase linked to the naturally occurring Cys102 near the C-terminus of yeast cytochrome c. This complex exhibits undetectable rates of intramolecular electron transfer. Complex II has Cys290 of the peroxidase linked to the engineered Cys73 of cyt c. This complex was designed to mimic the crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier & Kraut (1992) Science 258, 1748-1755]. Stopped-flow studies show that complex II carries out intramolecular electron transfer from ferrocytochrome c to peroxidase compound I at a rate of approximately 500-800 s-1. This indicates that the binding orientation observed in the crystal structure is competent in rapid intramolecular electron transfer.

A step towards understanding the folding mechanism of horseradish peroxidase. Tryptophan fluorescence and circular dichroism equilibrium studies.

The guanidinium chloride denaturation/renaturation of the holo- and apo-horseradish peroxidase isoenzyme c (HRP) has been studied by fluorescence and circular dichroism spectroscopies. A distinct equilibrium intermediate of the apoprotein could be detected at low concentrations of guanidinium chloride (0.5 M). This intermediate has a secondary structure content like that of the native protein but a poorly defined tertiary structure. Renaturation of the apo-HRP is reversible and 100% activity could be obtained after addition of a twofold excess of free haem. The denaturation of the holo-HRP is more complex and occurs in two distinct steps; unfolding of the protein backbone and loss of the haem. The denatured protein folds back to its native conformation but the incorporation of the haem occurs only after the secondary structure is formed. Ca2+ appears to be important for the stability of the protein as the apo-HRP is more resistant to denaturation in the presence of Ca2+. The free-energy change during unfolding of the apo-HRP was determined in the absence and presence of Ca2+ and found to be 9.2 kJ/mol and 16.7 kJ/mol, respectively. The importance of Ca2+ to the protein stability was also supported by studies on the loss of the haem from the protoporphyrin-IX-apo-HRP complex.