Several classes (chemical families) of herbicides are known to block photosystem II dependent electron transport at the level of the thylakoid membrane. The specificity of these herbicides for photosystem II makes them potentially powerful probes for elucidating the structure of the photosystem II complex. The mode of action of two such inhibitors, atrazine (a triazine herbicide) and diuron (a urea herbicide) have been extensively characterized; in both cases, these inhibitors act to block electron flow on the reducing side of photosystem II. Recently, K. Pfister, K. E. Steinback and C. J. Arntzen (Proc. Natl. Acad. Sci. USA, in press) have shown that a thylakoid membrane polypeptide with an apparent molecular weight between 32,000-34,000 becomes strongly labeled when stroma free thylakoids are incubated in the light with ¹⁴C-azido atrazine, indicating that a polypeptide in this size range serves as at least a portion of the atrazine binding site, and further suggesting that this polypeptide is functionally associated with electron transport components located on the reducing side of photosystem II. We have previously reported that thylakoids isolated from the green photosystem II deficient maize mutant hcf*-3 exhibit the nearly specific loss of a thylakoid membrane polypeptide with an apparent molecular weight of 32,000, and we have hypothesized on the basis of electrophoretic and ultrastructural data that this polypeptide may be required for the assembly of (preexisting?) components of the photosystem II reaction center (K. Leto and C. D. Miles, Plant Physiol. 66:18-24; K. Leto and C. J. Arntzen, Biochim. Biophys. Acta, in press; MGCNL 54:117-118). Since both lines of evidence (inhibitor binding and mutant studies) suggest that the 32,000 dalton polypeptide is a component of the photosystem II reaction center, we have examined the binding of both atrazine and diuron to stroma-free thylakoids prepared from hcf*-3 and wild type control seedlings.

The binding of ¹⁴C-atrazine and ¹⁴C-diuron (both uniformly ring labeled) was performed as described by Pfister, Radosevitch, and Arntzen (Plant Physiol. 64:995-999). Basically, the procedure involves incubation of a known quantity of stroma free thylakoid membranes (quantitated on a chlorophyll basis) with a known amount of ¹⁴C-labeled inhibitor, followed by centrifugation to separate the membranes and bound inhibitor from the "free" (unbound) inhibitor remaining in the supernatant. The amount of free inhibitor was quantitated by liquid scintillation spectrometry, and the amount of inhibitor bound to the thylakoids calculated by subtraction. A hyperbolic plot of the data (free inhibitor vs. inhibitor bound/chl) was used to measure saturation of high affinity binding sites; the number of binding sites on a chlorophyll basis (chl/binding site, X_i) and the apparent binding constant (Kb) were determined from double reciprocal plots.

The binding constants and the number of binding sites for both atrazine and diuron in thylakoids isolated from hcf*-3 and control seedlings were as follows:
 

	Wild Type		hcf*-3
Inhibitor	Kb	Xi	Kb	Xi
Atrazine	6.43 x 10-8	340	1.68 x 10-7	1800
Diuron	2.65 x 10-8	300	1.13 x 10-7	290

The values given for thylakoids obtained from control seedlings are in the range reported for other plant species similarly investigated. However, it is immediately apparent that the number of atrazine binding sites is greatly reduced in thylakoids prepared from hcf*-3 when compared to the number of sites observed in control thylakoids. In contrast, the number of diuron binding sites seen in thylakoids prepared from hcf*-3 was not reduced when compared to control membranes. Thylakoids prepared from hcf*-3 exhibited an increase in the binding constants for both atrazine and diuron, although the extent of this increase is small when compared to the extent of the loss of atrazine binding sites in these membranes. We conclude the following:

1) The loss of the 32,000 dalton lamellar polypeptide is accompanied by the parallel loss of photosystem II reaction centers and the loss of atrazine binding sites. Although alternative interpretations are possible, the data are consistent with the interpretations of Pfister et al. suggesting that a polypeptide in this size range is an integral part of the photosystem II complex and forms at least a portion of the atrazine binding site.

2) Although atrazine and diuron exhibit very similar modes of action at the level of the thylakoid membrane, the binding sites for the two are probably not identical, since thylakoids obtained from hcf*-3 contain a nearly normal number of diuron binding sites.

3) We have previously suggested that the 32,000 dalton thylakoid membrane polypeptide may be necessary for the organization of photosystem II, and that several of the polypeptides comprising the photosystem II reaction center complex may be present in an "unassembled" state in thylakoids obtained from hcf*-3. This hypothesis is supported by the discovery that thylakoids prepared from hcf*-3 contain a nearly normal number of diuron binding sites despite the loss of organized photosystem II reaction centers; diuron may be binding to photosystem II components which are present in these membranes but are not organized into functional reaction centers.

4) The observed increase in the herbicide binding constant for both atrazine and diuron in thylakoids isolated from hcf*-3 could be due to changes in the spatial orientation of the remaining photosystem II polypeptides, both with respect to each other and with respect to the lipid phase of the membrane.

Kenneth Leto, Klaus Pfister* and Charles Arntzen**

Present addresses: *Institut fur Botanik, University of Wurzburg, West Germany

**Director, MSU/DOE Plant Research Laboratory, Michigan State Univ., E. Lansing, MI 48824

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