This investigation will determine rate constants for proton transfer to and from bridging and terminal oxo and sulfide ligands in metalloproteins and model systems. By comparing the rate of reactivity of enzymes in H20 vs D20 we hope to establish fraction factors (preference for deuterium over hydrogen) and proton inventories (the number of protons involved in the rate-determining step). These studies should tell us which metalloproteins show slow proton transfer to or from oxygen or sulfur. Because of experimental limitations in protein systems (they cannot withstand large amount of acid or base, or high temperatures), systems which model different metalloprotein active sites will also examined. An understanding of these protonation reactions in model systems will shed light on the important but poorly understood reaction mechanisms of many metalloenzymes. Metalloproteins which contain oxo or sulfide bridges range from hemerythrin through ribonucleotide reductases and methane monooxygenase to the ferredoxin and high potential redox proteins. We will examine the reduction of iron center in the R2 unit of ribonucleotide reductase, and the catalytic activity of purple acid phosphatase and manganese catalase (which take part in many biological processes such as photosynthesis). The significance of protonations in such mechanisms is emphasized by the recent discovery that the intrinsic barrier (i.e. barrier at thermoneutrality) to protonation at bridging oxo ligands is much larger than at an ordinary oxygen. This study will determine if this result is general for all types of systems including biological ones, and compare the rates of protonation of oxo bridges with those of sulfide bridges and of terminal oxo and sulfide ligands.
