The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).

Iron is practically ubiquitous across Earth’s environments, central to both life and geochemical processes, which depend heavily on the redox state of iron. Although iron oxidation, or “rusting,” can occur abiotically at near-neutral pH, we find neutrophilic iron-oxidizing bacteria (FeOB) are widespread, including in aquifers, sediments, hydrothermal vents, pipes, and water treatment systems.

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples isolated using Triton X-100 detergent and samples further purified by n-heptyl beta-D-thioglucoside detergent treatment to remove light-harvesting polypeptides and low-affinity calcium. The structure of the manganese complex is very similar for the two detergent preparations used. Analysis of the e.x.a.f.s. spectra for samples in the S1 and S2 states indicate changes in bond lengths for the shells of oxygen/nitrogen atoms. For the S1 state, oxygen shells at 0.181 and 0.193 nm (1.81 and 1.93 A) were observed and one manganese shell at 0.270 nm (2.70A). In the S2 state the oxygen bond lengths are longer at 0.184 and 0.200 nm (1.84 and 2.00 A). Additionally a shell of scatterers at 0.37 nm (3.7 A) was observed in both states which could be fitted to models with calcium scatterers at this distance.

SummaryFe oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our ability to track the contributions of Fe-oxidizing microbes is limited by our relatively incomplete knowledge of microbial Fe oxidation mechanisms, particularly in neutrophilic Fe-oxidizers. The genomes of many Fe-oxidizers encode homologs to an outer-membrane cytochrome (Cyc2) that has been shown to oxidize Fe in two acidophiles. Here, we demonstrate the Fe oxidase function of a heterologously expressed Cyc2 homolog derived from a neutrophilic Fe oxidizer. Phylogenetic analyses show that Cyc2 from neutrophiles cluster together, suggesting a common function. Sequence analysis and modeling reveal the entire Cyc2 family is defined by a unique structure, a fused cytochromeporin, consistent with Fe oxidation on the outer membrane, preventing internal Fe oxide encrustation. Metatranscriptomes from Fe-oxidizing environments show exceptionally high expression of cyc2, supporting its environmental role in Fe oxidation. Together, these results provide evidence that cyc2 encodes Fe oxidases in diverse Fe-oxidizers and therefore can be used to recognize microbial Fe oxidation. The presence of cyc2 in 897 genomes suggests that microbial Fe oxidation may be a widespread metabolism.

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