Aconitase ENDOR

Figure 10. Inset, EPR absorption derivative spectnim of photoreduced active aconitase in the presence of citrate. Field positions at which ENDOR spectra were recorded are uvhcated by arrows. Numbm at the top are from the g-vdue scale. Main figure, 1 0 ENDOR spectrum at g = 1.88 for photoreduced active aconitase in the presence of citrate and 38% enriched (Reproduced with permission...

Table IV. EPR and ENDOR Results on Aconitase with 1 0 Labeled Substrate,...

Table V. ENDOR Results on Aconitase with f O Labeled Carboxylates of Substrates and Analogue...

The details obtained fiom the ENDOR work, in addition to past results, allow for the following mechanism for aconitase. In Scheme II the R group on the substrate is CH2COO, -B represents the amino acid side-chain which stereospeci-fically transfers a proton between citrate and isocitrate, and X is either water or a protein ligand. In the presence of c/s-aconitate, bound water can freely exchange... 

Figure 11. ENDOR spectra at g = 1.85 of photoreduced active aconitase in the presence of substrate whose carboxyl groups were individually labeled with...

In the absence of a 3-dimensional crystal strodure a rathd detailed picture of the active site of aconitase has been generated by the application of a variety of qrectro-scopic techniques. Most important of these has been the melding of informadmi from EPR, Mdssbauer and ENDOR spectroscopy. The future determination of the... 

Notwithstanding these studies, the role of the iron cluster in the catalysis remains an enigma. Clearly, the substrate binds to the Fca site by the Mdssbauer spectra. Also, the ENDOR spectra of aconitase with specifically 0-labeled substrates and inhibitors imply that the )3-carboxyl... 

Questions of detailed mechanism for aconitase remain open. ENDOR spectroscopy shows that both substrate and water (or OH ) can bind at the cluster. Does one of its Fe-atom vertices play the Lewis-acid role necessary for aconitase activity Is the Fe3S4 Fe4S4 conversion a redox- or iron-activated switch, which works as a control system for the activity of aconitase These and other questions will continue to be asked. If aconitase is indeed an Fe-S enzyme with an iron-triggered control mechanism, it may be representative of a... 

Aconitase was initially crystallized in the [3Fe-4S]" " form, and these crystals could be converted to the [4Fe S] " " form by addition of ferrous ammonium sulfate. Alternatively, anaerobic crystallization methods were used to crystallize the [4Fe-4S] " " form directly." The iron-sulfur cluster of aconitase sits within a solvent-filled cleft in the protein structure and is coordinated by Cys 358, Cys 421, and Cys 424. The [3Fe S]+ cluster shows the typically cuboidal geometry, with the open iron site directed toward the active site cleft. Conversion to the active [4Fe-4S] " " form results in almost no structural perturbation, with the extra iron atom simply being inserted into the vacant site in the cluster. The fourth iron is tetrahedrally coordinated, but with a solvent hydroxide rather than a cysteine as the fourth ligand. (The electron density clearly reveals a bound solvent, and previous ENDOR studies had demonstrated that the bound species was associated with only a single proton.)... 

Figure 4 Citrate binding to the active site [4Fe-4S] cluster of aconitase, as deduced from ENDOR...

Fig. 2. Representation of ENDOR-derived information about the [4Fe-4S] cluster of aconitase with substrate bound. As indicated, ENDOR studies showed four inequivalent Fe sites and two pairs of atoms. Exchangeable protons and O from bound hydroxide or water, and O and C from bound substrate, also were observed.

Fig. 15. O X-band cw ENDOR spectra at g = 1.85 (3815 G) of reduced aconitase in the presence of substrate whose -COO groups have been individually labeled with O (top) Label at a-carboxyl, (middle) label at /3-carboxyl, and (bottom) label at y-carboxyl. In the middle trace the bar indicates transitions centered at A/2 (filled circle) and split by 2i ( 0) (bars). (Adapted from Kennedy et al )...

One example of this method is for the protein aconitase, a 4Fe-4S protein that catalyzes the conversion of citrate to isocitrate. A complete cw ENDOR study was undertaken of this protein (see Fig. 2). Substrate binding information was obtained using citrate, and other substrates, labeled with 0 at specific positions. As shown in Fig. 15, this enabled the determination that citrate was bound to the Fe-S cluster through the center carboxylate group. This and other information determined by ENDOR led to a full description of the mechanism for aconitase. 

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