X-ray crystallographic investigations of the structure and function of oxidoreductases : a dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in the Institute of Fundamental Sciences, Chemistry, at Massey University, New Zealand
The structure and function of oxidoreductases were studied using the manganese-containing superoxide dismutase of Escherichia coli as the model system. The technique of single-crystal X-ray crystallography was used to determine the three-dimensional structure of this system. The structures of derivatives of this system, including iron-substituted manganese superoxide dismutase, and the five mutants Y34F, Q146H, Q146L, H30A and Y174F, were also determined. Analysis of these structures on a near-atomic scale revealed new structural aspects to the catalytic mechanism of this group of enzymes. A structural basis for the inactivity of E. coli Fe-substituted MnSOD has been determined in the altered geometry of the metal site on substitution of the non-native metal. The change in geometry from active five-coordinate trigonal-bipyramidal to inactive six-coordinate distorted octahedral modifies both the kinetics and thermodynamics of superoxide dismutation at the enzyme's metal centre. Gln146 is not essential for activity, but has an important role in optimising the reaction. Unlike the naturally active Hisl46-containing MnSOD enzymes, the mutation of E. coli MnSOD Glnl46 to histidine largely inactivates the enzyme. The inactivity may be a consequence of the greater inflexibility of the mutated histidine when compared with its natural counterparts. The lack of any change in both the primary and secondary coordination shells of the H30A mutant active-site, coupled with a 70 % reduction in catalytic activity, indicate an important role for His30 in optimising the catalytic mechanism. It is likely that the 60 % reduction in catalytic activity of the Y174F mutant is due to a different orientation, and possibly different effective pKa of His30, although a loss of activity due to the slight differences of the primary and secondary coordination spheres can not be entirely ruled out. Structural evidence supports a role for Tyrl74 in orienting and possibly also modifying the pK of His30. The association of His30, in particular via its ND1 nitrogen, with aspects of the catalytic mechanism including interaction or protonation of substrate, can be postulated based on its structural behaviour.