Genetic analysis of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 reveals subunit roles in association, assembly, maturation, and function

Abstract

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

FIGURE 1.

FPLC and Western blot analysis of WT Synechocystis sp. PCC 6803 Hox hydrogenase. The crude soluble fraction of WT Synechocystis sp. PCC 6803 was separated by FPLC, and the resulting fractions were analyzed by Western blotting. A, silver-stained TGX Any kDa gradient SDS-polyacrylamide gel (Bio-Rad) of starting material (Sol. Extr.) and fractions 1–22 (for the chromatogram at A₂₈₀ and A₄₂₀ see supplemental Fig. 1). B–G, Western blotting of FPLC fractions with the indicated Hox subunit-specific antibodies. Arrowheads in F indicate the signal for unprocessed HoxH.

FIGURE 2.

Two-dimensional BN/SDS-PAGE and Western blot analysis of WT Synechocystis sp. PCC 6803 Hox hydrogenase. A, the soluble fraction of WT Synechocystis sp. PCC 6803 was separated on a 12% (w/v) polyacrylamide BN-polyacrylamide gel followed by two-dimensional separation on a 17.5% (w/v) polyacrylamide SDS-polyacrylamide gel (B) and analysis by Western blotting with the indicated Hox subunit-specific antibodies (C–G). The first dimension BN-polyacrylamide gel was Coomassie-stained, whereas the second dimension SDS-polyacrylamide gel was silver-stained.

FIGURE 3.

One-dimensional SDS-PAGE and Western blot analysis of Synechocystis sp. PCC 6803 hox mutants. A, whole cell lysates of WT and individual/combined hox mutants were run on TGX Any kDa gradient SDS-polyacrylamide gels (Bio-Rad), transferred to PVDF, and immunoblotted with Hox subunit-specific antibodies and PsaD and/or Rps1 (not shown) as loading controls. B, relative levels of Hox subunits in each mutant, presented as a percentage of WT. Data represent quantitation of multiple Western blot analyses with error bars depicting variation between separate analyses.

FIGURE 4.

Two-dimensional-BN/SDS-PAGE and Western blot analysis of Synechocystis sp. PCC 6803 hox mutants. Shown are two-dimensional immunoblots (as described in the legend to Fig. 2) for hoxE⁻ (A), hoxF⁻ (B), hoxU⁻ (C), hoxY⁻ (D), and hoxH⁻ (E) deletion mutant strains probed with the indicated Hox subunit-specific antibodies.

FIGURE 5.

His₆ pull-down of Synechocystis sp. PCC 6803 Hox subcomplexes. hox⁻ mutant expressing His₆-HoxYH (A), hoxH⁻ mutant expressing His₆-HoxH (B), and hoxE⁻ mutant expressing His₆-HoxE (C) were used for pull-down on Co²⁺ beads to determine subunit association. Supernatant, flow-through (FT) following bead incubation, pooled washes with/without 0.5 m NaCl, and boiled bead samples from each pull-down were run on TGX Any kDa gradient SDS-polyacrylamide gels (Bio-Rad), transferred to PVDF, and immunoblotted using Hox subunit-specific antibodies.

FIGURE 6.

Relative hydrogenase activity of Synechocystis sp. PCC 6803 hox mutants. Whole cells of Synechocystis sp. PCC 6803 WT and hox⁻ mutant strains were analyzed for hydrogenase activity with the exogenous electron donor MV. The graph shows MV-linked activities of hox mutants relative to WT with assay variation depicted by error bars. Rates (nmol of H₂ ml⁻¹ min⁻¹) are presented in supplemental Table 1.

FIGURE 7.

Fluorescence-based ETR measurements for Synechocystis sp. PCC 6803 WT and hox⁻ strains grown under 50 μE m⁻² s⁻¹ continuous illumination. Variable fluorescence parameters of dilute whole cell suspensions were collected after 3 min of actinic light adaptation at varying intensities. ETR = ΔF/Fm′ × (actinic light intensity) × 0.42. Error bars represent standard deviations of three biological replicates.

FIGURE 8.

Model for Hox hydrogenase association in Synechocystis sp. PCC 6803. Unprocessed HoxH (HoxH*; containing an additional 16-amino acid C terminus) binds HoxY (containing a [4F-4S] cluster) and undergoes assembly and insertion of its [NiFe] active site and cleavage of its C terminus by the protease HoxW, resulting in a hydrogenase subcomplex (HoxYH). Alternatively, HoxF (containing NAD and FMN binding sites and [2Fe-2S] and [4Fe-4S] clusters) associates with HoxU (containing one [2Fe-2S] and three [4Fe-4S] clusters) and HoxE (containing a [2Fe-2S] cluster), forming the diaphorase subcomplex (HoxEFU). The presence and formation of HoxFU and HoxFUYH subcomplexes may represent steps in assembly, but may also formally be due to a loss of HoxE during preparation/extraction strategies. Purification by Schmitz et al. resulted in detection of a dimer of the HoxEFUYH pentamer ((HoxEFUYH)₂), which is depicted here despite our inability to detect this dimer in our preparations. We hypothesize that this dimerization may be through HoxH, allowing association of unprocessed HoxH with the full complex.