Curtis W. Hoganson

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution

Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase I. BIOCHEMICAL, SPECTRAL, AND KINETIC CHARACTERIZATION OF SURFACE MUTANTS IN SUBUNIT II OF RHODOBACTER SPHAEROIDESCYTOCHROME aa 3

To determine the interaction site for cytochromec (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp143, was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc2, showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K m values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp143 severely inhibit activity without changing theK m for Cc or disturbing the adjacent CuA center. From these data, we identify a Cc binding area on CcO with Trp143 and Asp214 close to the site of electron transfer and Glu148, Glu157, and Asp195 providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem.274, 38042–38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051–38060).

Mn2+ reduces Yz+ in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn2+. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Yz+, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Yz+ reduction by benzidine was a linear function of benzidine concentration. The rate of Yz+ reduction by Mn2+ at pH 6 increased linearly at low Mn2+ concentrations and reached a maximum at the Mn2+ concentrations equal to several times the reaction center concentration. The rate was inhibited by K+, Ca2+ and Mg2+. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Yz+ reduction at pH 7.5 was biphasic with a fast 400 μs phase that suggests binding of Mn2+ near Yz+ at a site that may be one of the native manganese binding sites.

Manganese and tyrosyl radical function in photosynthetic oxygen evolution

Photosystem II catalyzes the photosynthetic oxidation of water to O2. The structural and functional basis for this remarkable process is emerging. The catalytic site contains a tetramanganese cluster, calcium, chloride and a redox-active tyrosine organized so as to promote electroneutral hydrogen atom abstraction from manganese-bound substrate water by the tyrosyl radical. Recent work is assessed within the framework of this model for the water oxidizing process.

From water to oxygen and back again: mechanistic similarities in the enzymatic redox conversions between water and dioxygen.

We propose that the interconversions of water and oxygen are catalyzed by the transition metal ions of Photosystem II and cytochrome c oxidase in remarkably similar ways. Oxygen-oxygen bond formation and cleavage occurs between two oxygen atoms that are bound as terminal ligands to two redox-active metal ions. Hydrogen atom transfer to or from a tyrosine residue is an essential component of the processes in both enzymes.

The Role of Magnesium and Its Associated Water Channel in Activity and Regulation of Cytochrome cOxidase

Research on cytochrome coxidase has moved into a new era with the recent resolution of the crystal structures of bacterial [1] and beef heart [2] enzymes, showing their remarkable similarity. The resolution of the crystal structures has specified the spatial organization of the metal centers and defined some possible routes for proton translocation within the molecule. Three distinct pathways for protons are expected in the cytochrome coxidase: two entries for the pumped and substrate protons and an unidirectional exit route. An apparent water channel, which could function as the proton exit pathway, is clearly visible in the beef heart oxidase X-ray structure [2] (Figure 1): it is immediately above the active site and connects it to the exterior of the membrane.

Electron and Proton Transfer in Heme-Copper Oxidases

In eukaryotes, the process of energy transduction coupled to electron transfer occurs in mitochondria, where cytochrome c oxidase catalyzes the transfer of electrons derived from foodstuffs to oxygen, the final electron sink. The reduction of oxygen to water and the concomitant translocation of protons are carried out by a member of a family of enzymes: the heme-copper oxidases (Saraste, 1990; Garcia-Horsman et al., 1994). A number of these oxidases have now been identified in bacterial systems; all use a heme-copper center to carry out the oxygen chemistry, but not all have similar auxiliary metal centers. The eu karyotic and some prokaryotic enzymes contain an additional heme a, an additional bimetallic copper center, and a magnesium ion. These oxidases use cytochrome c as their immediate electron donor. Another group of oxidases in the family use quinol as a substrate and contain neither an extra copper center nor magnesium. Although the two groups are likely to have similar energy transduction mechanisms, this discussion will focus on the cytochrome c oxidases with the additional copper and magnesium and on our efforts to define the roles of these auxiliary metals in controlling electron input, proton output, and coupling efficiency.

The proton enviroment about Yz as a function of metal content in photosystem II

Recent work indicates that YZ and the manganese cluster in Photosystem II form an integrated catalytic site for water oxidation (1–4). This realization, coupled with the idea that the function of amino-acid radicals in the emerging class of radical enzymes is to abstract hydrogen atoms from substrate, leads to a complete model for the S-state cycle for water oxidation in photosynthesis (5,6, see Figure 1). A key aspect of this mechanism is that, upon its oxidation, YZ sloughs a proton and that this H+ is released to bulk phase upon each of the S-state transitions. Although fast proton release is detected upon each S-state transition (7), the origin of this proton release remains controversial (5,6,8–10). Here, we summarize recent work that indicates that electro-neutrality is maintained at the YZ site upon each S-state transition and that provides insight into the accessibility of the YZ site to bulk solvent as a function of the integrity of the (Mn)4/Ca site in the oxygen-evolving complex.

Analytical procedures for the quantification of isotopic amino acid incorporation into photosynthetic proteins of Synechocystis PCC 6803.

The mechanism of oxygen evolution has been an enigma for nearly two centuries. Pioneering work by Bessel Kok, Pierre Joliot, and many others during the last quarter century has provided valuable insight into this most unique and important chemical reaction. The late 1970s and early 1980s saw the introduction of biochemical techniques for the purification of photosynthetic complexes that have, in turn, stimulated the biophysical chemists and spectroscopists to apply high resolution techniques in order to resolve the structure/function relationships in these protein complexes. Valuable information about events at the atomic level can be gained through isotopic substitution of particular amino acids thought to be important in the catalytic process. The ability to generate functional auxotrophs in the photosynthetic cyanobacterium Synechocystis 6803 has been used successfully to identify the redox active components Z and D as tyrosine residues in the reaction center of Photosystem II. In this report, we present results of the application of specific isotopic labeling for high resolution spectroscopy of purified PS II particles. We have developed analytical procedures for monitoring the incorporation of both 2H and 17O labeled amino acids by gas chromatography-mass spectroscopic analysis. We also show that the growth curve of cells subjected to obligate auxotrophy displays two distinct stationary phases; one that corresponds to depletion of exogenous amino acids, and a second that corresponds to the normal cell density at stationary phase. Cells harvested at the second stationary phase show little or no retention of the labeled amino acid.