Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn21 or Cd21 (bacterial photosynthesisyRhodobacter sphaeroidesymetal bindingyproton-coupled electron transfer)

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB (QAQB 1 H1 3 QA(QBH), where QA is the primary quinone acceptor). Zn21 and Cd21 bound stoichiometrically to the RC (KD < 0.5 mM) and reduced the observed value of kAB 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB reaction. In native RCs, kAB was previously shown to be rate-limited by electron transfer based on the dependence of kAB on the driving force for electron transfer. Upon addition of Zn21 or Cd21, kAB became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (> 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction DQAQB3 DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB occurs. The bacterial reaction center (RC) is a membrane-bound pigment protein complex that converts light into chemical energy through a two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor) to a quinol molecule QBH2 (1, 2). The protons taken up to form quinol are transferred from the solvent of the cytoplasm to the bound quinone molecule. Understanding the details of proton transfer in this and other systems is important for a basic understanding of bioenergetics. This paper addresses the pathway of the proton transfer by measuring the effects of metal ion binding on proton transfer rates in the bacterial RCs from Rhodobacter sphaeroides. The isolated RC is composed of three polypeptide subunits (L, M, and H), four bacteriochlorophylls, two bacteriopheophytins, one internally bound nonheme Fe21, and two ubiquinone (UQ10) molecules (reviewed in refs. 1 and 2). In the RC, the light-induced electron transfer proceeds from a primary donor (a bacteriochlorophyll dimer) through a series of electron donor and acceptor molecules (a bacteriopheophytin and a primary quinone acceptor QA) to a loosely bound secondary quinone QB, which serves as a mobile electron and proton carrier (3–5), transferring electrons and protons from the RC to other components of the bioenergetic cycle. The first electron transfer to QB (kAB) does not involve direct protonation of the quinone (Eq. 1).