Spectroscopic and Functional Characterizations of Cyanobacterium Synechocystis PCC 6803 Mutants on and near the Heme Axial Ligand of Cytochrome b559 in Photosystem II

Hong Jin Hwang,Yi-Fang Chiu,Chung-Hsien Hung,Robert L Burnap,Yung-Han Chen,Hsiu-An Chu,Shyue-Chu Ke

doi:10.1074/jbc.m109.044719

Abstract

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Highlights

PSII reaction centers, and show the normal period-four oscillation in oxygen yield. Both Y18S␣ and H22K␣ cyt b559 mutant PSII core complexes retained the heme of cyt b559 and gave rise to distinct optical absorption and EPR signals of cyt b559
Our results showed that H22K␣ mutant PSII core complexes gave rise to EPR signals, which are characteristic for the high spin form of cyt b559
These EPR signals were observed in R7L␣ mutant PSII core complexes [36] and in PSII membranes treated with 2,3-dichloro-5,6-dicyano-p-benzoquinone [37]

Summary

EXPERIMENTAL PROCEDURES

Experimental conditions for measurements of PSII fluorescence yield in the presence and absence of actinic light and decay of flash-induced variable fluorescence in response to a saturating flash given to wild-type and mutant cells are described in the legend to Fig. 1. Reduced Minus Oxidized Difference Spectra of cyt b559 in Wild-type and Mutant PSII Core Complexes—Optical absorption difference measurements were performed on suspensions of wild-type* and mutant PSII core complexes (20 ␮g/ml Chl) in 1 ml of MMNB buffer (25 mM Mes, 5 mM MgCl2, 10 mM NaCl, 1 M glycine betaine, 0.03% n-dodecyl ␤-D-maltoside, pH 5.7) at room temperature on a Jasco V-560 UV-visible spectrophotometer with a bandwidth of 1.0 nm.

Wild type ϩ

RESULTS

Redox formsa

DISCUSSION