CRYSTAL-STRUCTURE OF Y34F MUTANT HUMAN MITOCHONDRIAL MANGANESE SUPEROXIDE-DISMUTASE AND THE FUNCTIONAL-ROLE OF TYROSINE-34

Tyrosine 34 is a prominent and conserved residue in the active site of the manganese superoxide dismutases in organisms from bacteria to man . We have prepared the mutant containing the replacement Tyr 34 --> Ph e (Y34F) in human manganese superoxide dismutase (hMnSOD) and crystall ized it in two different crystal forms, orthorhombic and hexagonal. Cr ystal structures of hMnSOD Y34F have been solved to 1.9 Angstrom resol ution in a hexagonal crystal form, denoted as Y34F(hex), and to 2.2 An gstrom resolution in an orthorhombic crystal form, denoted as Y34F(ort ho). Both crystal forms give structures that are closely superimposabl e with that of wild-type hMnSOD, with the phenyl rings of Tyr 34 in th e wild type and Phe 34 in the mutant very similar in orientation. Ther efore, in Y34F, a hydrogen-bonded relay that links the metal-bound hyd roxyl to ordered solvent (Mn-OH to Gin 143 to Tyr 34 to H2O to His 30) is broken. Surprisingly, the loss of the Tyr 34 hydrogen bonds result ed in large increases in stability (measured by T-m), suggesting that the Tyr 34 hydroxyl does not play a role in stabilizing active-site ar chitecture. The functional role of the side chain hydroxyl of Tyr 34 c an be evaluated by comparison of the Y34F mutant with the wild-type hM nSOD. Both wild-type and Y34F had k(cat)/K-m near 10(9) M-1 s(-1), clo se to diffusion-controlled; however, Y34F showed k(cat) for maximal ca talysis smaller by 10-fold than the wild type. In addition, the mutant Y34F was more susceptible to product inhibition by peroxide than the wild-type enzyme. This activity profile and the breaking of the hydrog en-bonding chain at the active site caused by the replacement Tyr 34 - -> Phe suggest that Tyr 34 is a proton donor for O-2(.-) reduction to H2O2 or is involved indirectly by orienting solvent or other residues for proton transfer. Up to 100 mM buffers in solution failed to enhanc e catalysis by either Y34F or the wild-type hMnSOD, suggesting that pr otonation from solution cannot enhance the release of the inhibiting b ound peroxide ion, likely reflecting the enclosure of the active site by conserved residues as shown by the X-ray structures. The increased thermostability of the mutant Y34F and equal diffusion-controlled acti vity of Y34F and wild-type enzymes with normal superoxide levels sugge st that evolutionary conservation of active-site residues in metalloen zymes reflects constraints from extreme rather than average cellular c onditions. This new hypothesis that extreme rather than normal substra te concentrations are a powerful constraint on residue conservation ma y apply most strongly to enzyme defenses where the ability to meet ext reme conditions directly affects cell survival.