Eui-Jin Kim

Superoxide Generation by Chlorophyllide a Reductase of Rhodobacter sphaeroides

Chlorophyllide a reductase of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides Fe-SOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of chlorophyllide a reductase. The enzyme is labile in the presence of O2. However, it generates superoxide at low O2. The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half-reduced in its redox state compared with that of wild-type BchZ.

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products

BackgroundBiological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium.ResultsAs continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD+ and NADP+. In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control.ConclusionsWe have found that SMPs are the key factor in photo-fermentative H2 production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR‐mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S‐adenosylmethionine‐limiting conditions

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O2‐dependent HemF and O2‐independent HemN. The growth of the hemF mutant HF1 was similar to wild‐type cells at pH 7.5 under 2% O2 conditions where HemN was active and had a half‐life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S‐adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM‐limiting conditions, a mutation arose in hemN, encoding HemNY74F. Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemNY74F for the cofactor SAM1, which coordinates the 4Fe‐4S cluster, was approximately sixfold higher than that of HemN. The Km of HemNY74F for the co‐substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.