THE AXIAL TYROSINATE FE3-DIOXYGENASE INFLUENCES SUBSTRATE-BINDING ANDPRODUCT RELEASE - EVIDENCE FOR NEW REACTION CYCLE INTERMEDIATES( LIGAND IN PROTOCATECHUATE 3,4)
Rw. Frazee et al., THE AXIAL TYROSINATE FE3-DIOXYGENASE INFLUENCES SUBSTRATE-BINDING ANDPRODUCT RELEASE - EVIDENCE FOR NEW REACTION CYCLE INTERMEDIATES( LIGAND IN PROTOCATECHUATE 3,4), Biochemistry, 37(8), 1998, pp. 2131-2144
The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3,4
-PCD, subunit structure (alpha beta Fe3+)(12)] is bound by axial ligan
ds, Tyr447 (147 beta) and His462 (162 beta), and equatorial ligands, T
yr408 (108 beta), His460 (160 beta), and a solvent OH- (Wat827). Recen
t X-ray crystallographic studies have shown that Tyr447 is dissociated
from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (
PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Bio
chemistry, 36, 10052-10066]. The importance of Tyr447 to catalysis is
investigated here by site-directed mutation of this residue to His (Y4
47H), the first !;uch mutation reported for an aromatic ring cleavage
dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 Angst
rom resolution, R-factor of 0.181) is essentially unchanged from that
of the native enzyme outside of the active site region. The side chain
position of His447 is stabilized by a His447N(N delta 1)-Pro448(omicr
on) hydrogen bond, placing the N epsilon 2 atom of His447 out of bondi
ng distance of the iron (similar to 4.3 Angstrom). Wat827 appears to b
e replaced by a CO32-, thereby retaining the overall charge neutrality
and coordination number of the Fe3+ center. Quantitative metal and am
ino acid analysis shows that Y447H binds Fe3+ in similar to 10 of the
12 active sites of 3,4-PCD, but its k(cat),,, is nearly 600-fold lower
than that of the native enzyme. Single-turnover kinetic analysis of t
he Y447H-catalyzed reaction reveals that slow substrate binding accoun
ts for the decreased k(cat),,,. Three new kinetically competent interm
ediates in this process are revealed. Similarly, the product dissociat
ion from Y447H is slow and occurs in two resolved steps, including a p
reviously unreported intermediate. The final E.PCA complex (ES4) and t
he putative E.product complex (ESO2) are found to have optical spectr
a that are indistinguishable from those of the analogous intermediates
of the wild-type enzyme cycle, while all of the other observed interm
ediates have novel spectra. Once the E.S complex is formed, reaction w
ith O-2 is fast. These results suggest that dissociation of Tyr447 occ
urs during turnover of 3,4-PCD and is important in the substrate bindi
ng and product release processes. Once Tyr447 is removed from the Fe3 in the final E.PCA complex by either dissociation or mutagenesis, the
O-2 attack and insertion steps proceed efficiently, suggesting that T
yr447 does not have a large role in this phase of the reaction. This s
tudy demonstrates a novel role for Tyr in a biological system and allo
ws evaluation and refinement of the proposed Fe3+ dioxygenase mechanis
m.