Analysis of the OmcZ sequence.
The omcZ gene (GSU2076; GenBank accession no. AAR35452) encodes a protein of 473 amino acids with 7 typical heme c-binding motifs (CXXCH), as well as the possible heme-binding sequence CX14CH (Fig. ). The program PSORT predicted OmcZ to be localized in the outer membrane (score, 0.39; certainty, 0.743). The topology prediction programs SOSUI and TMHMM suggested that the N-terminal region (amino acids 5 to 27) forms a transmembrane helix. According to the SignalP server 3.0, OmcZ has a signal sequence with a predicted cleavage site between amino acids 24 and 25 in the transmembrane helix. The cleaved polypeptide, which consists of 449 residues, contains no transmembrane site.
FIG. 1. Multiple alignment of OmcZ homologues. The identical, strongly similar, and weakly similar residues are indicated by asterisks, colons, and dots, respectively. The (putative) signal peptide cleavage sites are shown by dashed lines. The boxes represent (more ...)
No close OmcZ homologues from outside the genus Geobacter
were found in the GenBank database. The amino acid sequence of OmcZ had the following percent identities to Geobacter
proteins: 50.3 to GSU1334 from G. sulfurreducens
(GenBank accession no. AAR34710), 46.2 to Gbem_3056 from Geobacter bemidjiensis
strain Bem (ACH40058), 45.6 to GM21_1194 from Geobacter
sp. strain M21 (EDV70209), and 44.5 to Gmet_0930 from Geobacter metallireducens
strain GS-15 (ABB31172). The unusual heme-binding motif CX14
CH was completely conserved in GM21_1194 and Gbem_3056 and was also conserved with shorter distances between the cysteine residues in GSU1334 (CX13
CH) and Gmet_0930 (CX11
CH) (Fig. ). From amino acid 350 to the C terminus, OmcZ showed homology with the D domain of cyclodextrin glycosyltransferases from Bacillus
spp. The D domain is well conserved among the cyclodextrin glycosyltransferases, but the function of the domain is still unknown (46
Subcellular localization of OmcZ.
It was previously reported that the loosely bound outer membrane fraction from G. sulfurreducens
contained two forms of OmcZ with masses of approximately 50 and 30 kDa on SDS-PAGE gels (39
). The large and the small forms were designated OmcZL
OmcZS was more abundant than OmcZL in wild-type cells and in strain ZKI, which had been engineered to overexpress OmcZ (Fig. ). In wild-type cells, both forms of OmcZ were detected in the outer membrane fraction and culture supernatant, but only OmcZS was detected in the cell debris fraction, which primarily contained insoluble extracellular matrix material (Fig. ). More OmcZS was found in the supernatant and cell debris fractions of stationary-phase cells than in mid-log-phase cells. To evaluate the relative amounts of protein in the fractions, the signal intensities of the bands of the Western blots were multiplied by the total amount of protein in each fraction. This analysis indicated that OmcZS was most abundant in the cell debris fraction regardless of the growth phase (data not shown). When OmcZ was overexpressed in strain ZKI, some OmcZL was also detected in the periplasm of mid-log-phase cells, but OmcZS was not (Fig. ). In both mid-log- and stationary-phase cells, OmcZS was mainly distributed in the cell debris fraction and partially in the outer membrane fraction (Fig. ).
FIG. 2. Subcellular localization of OmcZL and OmcZS in G. sulfurreducens strains DL-1 and ZKI. (A) Western blot with anti-OmcZ antibody of subcellular fractions of mid-log- and stationary-phase wild-type cells and subcellular fractions of mid-log- and stationary-phase (more ...)
These results demonstrated that the mature protein OmcZS
can be found in the outer membrane and cell debris fractions but not in the periplasmic or cytoplasmic fraction. The recovery of some OmcZL
, but not OmcZS
, in the periplasm suggests that OmcZL
is cleaved during secretion across the outer membrane, as has been reported for the secretion of other outer-surface proteins in other microorganisms (55
Purification of OmcZS.
OmcZS was purified from the stationary-phase cells of strain ZKI because it was much more abundant than OmcZL in these cells (Fig. ). It was purified by detergent extraction followed by gel filtration chromatography. After detergent extraction, the protein sample was already purified to near homogeneity (Fig. ). The detergent-extracted OmcZS, originally solubilized in 10% Zwittergent 3-14 solution, was poorly soluble in biochemical buffers, such as 50 mM Tris-HCl (pH 7.0), after removal of the detergent. However, detergent-extracted OmcZS was readily soluble in pure water. Almost all of the protein stacked on top of the gel when the sample was loaded without heating (see Fig. S1A in the supplemental material). A clear OmcZS band appeared when the sample was heated at 100°C for 5 min with SDS sample buffer (Fig. ). Moreover, the detergent-extracted OmcZS was also retained on a 300-kDa-cutoff filter after centrifugation (see Fig. S1B in the supplemental material). These observations indicate that OmcZS polymerizes or assembles with other cell constituents at this stage. To disassemble the OmcZS into monomers, the detergent-extracted OmcZS was heated (90°C) with 1% SDS for 5 min. After this procedure, OmcZS passed through 300-kDa-cutoff filters (see Fig. S1B in the supplemental material).
FIG. 3. OmcZS expression (A), purification (B), and Western blot analysis (C). The proteins were separated by 12.5% Tris-Tricine denaturing polyacrylamide gel electrophoresis. (A) Heme-stained loosely bound outer membrane protein-enriched fractions from (more ...)
The heat-treated sample was further purified by gel filtration chromatography. The single peak for OmcZS was the only peak observed in the chromatograph (data not shown). The relative molecular mass of OmcZS was calculated as 32 kDa by gel filtration chromatography. Typical yields were approximately 2 mg of protein per liter of culture. After gel filtration chromatography, the purified OmcZS was poorly soluble in 50 mM Tris-HCl buffer but highly soluble in pure water. The purified OmcZS in water remained on the 300-kDa-cutoff filters after centrifugation again (see Fig. S1B in the supplemental material), indicating that OmcZS reassembled after removal of the detergents. SDS-PAGE analysis also showed self-assembling characteristics of OmcZS. The apparently dimerized and trimerized bands of the purified OmcZS were observed when the protein sample was not heated before being loaded on the SDS-PAGE gel (see Fig. S1A in the supplemental material). SDS-PAGE and Western blotting confirmed that the final product of the purification was pure and contained a single protein band with a mass of ca. 30 kDa (Fig. ).
Physical and chemical properties of OmcZS. (i) N and C termini of OmcZS, molecular mass, and heme content.
ESI-MS analysis indicated an average molecular mass of 32,582 Da for the purified OmcZS when measured in 50% methanol and 3% acetic acid and 32,578 Da when measured under milder conditions in 10 mM ammonium bicarbonate buffer. Edman sequencing analysis of the purified OmcZS revealed that the N terminus sequence is AVPPP, which is located 25 to 29 residues from the N terminus (Fig. ). This indicates that the N terminus signal peptide of OmcZ is cleaved between amino acids 24 and 25, which corresponds with the cleavage site predicted by SignalP.
Digestion of the OmcZS with carboxypeptidase Y yielded 3 amino acids: Gly (44% of the total amino acids detected [mol/mol]), Phe (33%), and Asn (19%). Among all possible sequence combinations of these 3 amino acids, GNF (136 to 138 from the N terminus) and FGN (280 to 282) were found in the OmcZ amino acid sequence. A protein with the latter C terminus gave the expected molecular mass of ca. 30 kDa.
The estimated molecular mass of OmcZS is consistent with the predicted amino acid content (Fig. ) and eight heme groups with a molecular mass of 616 Da. The predicted peptide sequence of OmcZS (Fig. ) contains all the seven predicted OmcZ heme-binding sites represented by the CXXCH motif, as well as the unusual (CX14CH) heme-binding site. Pyridine hemochrome analysis revealed that OmcZS contains 7.7 hemes per molecule, indicating that hemes bind to all the possible heme-binding sites.
In general, c
-type cytochrome biogenesis in bacteria has been thought to be strictly dependent on the presence of two cysteine residues arranged in a CX2-4
CH/K motif (16
). However, the octaheme c
-type cytochrome MccA in Wolinella suggincogenes
contains a covalent heme attached to an unusual heme-biding CX15
CH motif, which requires a specialized cytochrome c
heme lyase, CcsA1, for heme attachment (16
). Six CcsA1-type heme lyase homologues are in the G. sulfurreducens
), one or more of which could account for the heme incorporation into the unusual heme-binding site in OmcZ.
(ii) Thermal stability, circular dichroism (CD), and optical spectra.
OmcZS was exposed to heat (90°C) and harsh detergents, such as SDS and Zwittergent 3-14, in the purification procedure. DSC analysis of the thermal stability of OmcZS indicated that the denaturation temperature was 94.2°C at pH 7.0.
CD spectra of purified OmcZS demonstrated the presence of significant secondary structures. The far-UV CD spectrum indicated that OmcZS is comprised of 13% α-helix, 18% antiparallel β-sheet, 5% parallel β-sheet, and 28% β-turn.
The UV/visible redox spectrum was characteristic of c
-type cytochromes (Fig. ). The maxima of the spectrum of the oxidized OmcZS
were at 408 nm (910,200 M−1
; γ Soret band) and 530 nm (103,130 M−1
) (Fig. ). After reduction with DTT for 1 h, OmcZS
had absorption maxima at 419 nm (1,084,400 M−1
; shifted γ Soret band), 523 nm (126,000 M−1
; β Soret band), and 552 nm (171,000 M−1
; α Soret band). These spectra are typical for c
-type cytochromes with six coordinated low-spin hemes (2
UV/visible absorption spectra of OmcZS. The dotted and solid lines represent oxidized and reduced cytochrome, respectively.
(iii) Redox characteristics of OmcZS.
The redox behavior of OmcZs was investigated with redox titrations, followed by visible spectroscopy (Fig. ). Both the oxidative and reductive curves spanned a large range of reduction potentials (−420 to −60 mV). The curves exhibited some hysteresis, which indicates that under these experimental conditions the protein can cycle between the fully reduced and fully oxidized states in a nonreversible way. This suggests that slowly relaxing modifications in the protein structure are associated with the redox transition. The Eapp
(i.e., the point at which the oxidized and reduced fractions are equal) values for the reductive and oxidative curve were −206 and −234 mV (versus the standard hydrogen electrode [SHE]), respectively. The shapes of the experimental curves deviate significantly from one that considers identical reduction potential values for the 8 heme groups (dashed line in Fig. ). This observation points to nonequivalence of the redox centers, which is expected for a multiredox center protein with eight heme groups (47
), as is the case for OmcZS
FIG. 5. Redox titrations followed by visible spectroscopy for OmcZS at 298 K and pH 7. The open and filled symbols represent the data points in the reductive and oxidative titrations, respectively. The continuous lines indicate the fit to a model considering (more ...)
The large potential range of OmcZ (−420 to −60 mV versus SHE) can most probably be attributed to the wide range of the redox potentials for the 8 hemes in the molecule. This is similar to the decaheme c
-type outer-surface cytochrome MtrC of S. oneidensis
, which has a potential range of −500 to +100 mV (15
). The potential range of OmcZ covers the lowest anode potential observed in microbial fuel cells of G. sulfurreducens
(−420 mV versus Ag/AgCl, approximately equal to −220 mV versus SHE) (5
), suggesting that OmcZ has a low enough potential to directly transfer electrons to the anode.
(iv) Electron acceptors.
Spectrophotometric analysis revealed that OmcZS is rapidly (less than 5 min) oxidized with known electron acceptors for G. sulfurreducens, such as Fe(III) citrate, Mn(IV) oxide, U(VI), Cr(VI), Au(III), and the humic acid analogue AQDS (Fig. ). OmcZS was only partially reoxidized when it was incubated with Fe(III) oxide (Fig. ). Even after 90 min of incubation, the 419-nm γ-band did not completely shift to 408 nm (Fig. , inset), indicating that OmcZS has little activity toward Fe (III) oxide. After the addition of Fe(III) oxide, all the peaks decreased with time, probably because OmcZS attached to the insoluble Fe(III) oxide.
FIG. 6. Reduction of metals and AQDS with reduced OmcZS. Distilled water (dH2O) (negative control) (A), Fe(III) citrate (B), Mn(IV) (C), U(VI) (D), Cr(VI) (E), Au(III) (F), AQDS (G), and Fe(III) oxide (H) were added to reduced OmcZS. (A to G) The UV/visible spectra (more ...)
Previous studies have suggested that c
-type cytochromes are involved in the reduction of a variety of metals and the humic acid analogue AQDS by Geobacter
). The ability of OmcZ to transfer electrons to metals and AQDS, coupled with its extracellular location, suggests that OmcZ could play a role in electron transfer to these extracellular electron acceptors. The finding that OmcZ did not readily transfer electrons to insoluble Fe(III) oxide is consistent with the fact that deleting the gene for OmcZ did not have any impact on the capacity for Fe(III) oxide reduction by G. sulfurreducens
). OmcZ could reduce Mn(IV) oxide quickly, but not Fe(III) oxide. This might be explained by the fact that the midpoint potential of Mn(IV) oxide is much higher (from 500 to 600 mV at 25°C, pH 7 [54
]) than that of OmcZ (−220 mV), whereas the midpoint potential of Fe(III) oxide (−300 to 0 mV [54
]) is comparable to that of OmcZ.
Several of the characteristics of OmcZ reported here are consistent with the requirement for OmcZ for optimal current production by G. sulfurreducens
) and electrochemical results (48
) that suggest that OmcZ aids in electron conduction from G. sulfurreducens
biofilms to the anodes of microbial fuel cells. The results demonstrate that OmcZ is primarily localized in the extracellular matrix, as would be expected for a protein contributing to electron conduction in biofilms. Precedents for extracellular localization of c
-type cytochromes include the presence of the cytochromes MtrC and OmcA in the polymeric substance surrounding cells of S. oneidensis
) and recovery of a c
-type cytochrome in the extracellular matrix purified from Myxococcus xanthus
). The tendency for OmcZ to self-assemble, coupled with its poor solubility in buffers, suggests that OmcZ would be retained within the biofilm matrix rather than lost to the external medium. Therefore, the energy commitment to produce OmcZ to promote electron transfer through the biofilm is not likely to be dissipated in loss of OmcZ to the external medium. The multiple redox potentials of the hemes in OmcZ seem well suited to promoting electron transfer to anode biofilms, which may function at different potentials, depending on the resistance to electron flow and the rates of metabolism in microbial fuel cells. Studies are now under way to purify OmcS, another abundant outer-surface c
-type cytochrome of G. sulfurreducens
that is required for Fe(III) oxide reduction but not for high-density current production, in order to compare biochemical features that differentiate the functions of outer-surface cytochromes.