Formation of Proton Motive Force Under Low-Aeration Alkaline Conditions in Alkaliphilic Bacteria

In Mitchell’s chemiosmotic theory, a proton (H+) motive force across the membrane (Δp), generated by the respiratory chain, drives F1Fo-ATPase for ATP production in various organisms. The bulk-base chemiosmotic theory cannot account for ATP production in alkaliphilic bacteria. However, alkaliphiles thrive in environments with a H+ concentrations that are one-thousandth (ca. pH 10) the concentration required by neutralophiles. This situation is similar to the production of electricity by hydroelectric turbines under conditions of very limited water. Alkaliphiles manage their metabolism via various strategies involving the cell wall structure, solute transport systems and molecular mechanisms on the outer surface membrane. Our experimental results indicate that efficient ATP production in alkaliphilic Bacillus spp. is attributable to a high membrane electrical potential (ΔΨ) generated for an attractive force for H+ on the outer surface membrane. In addition, the enhanced F1Fo-ATPase driving force per H+ is derived from the high ΔΨ. However, it is difficult to explain the reasons for high ΔΨ formation based on the respiratory rate. The Donnan effect (which is observed when charged particles that are unable to pass through a semipermeable membrane create an uneven electrical charge) likely contributes to the formation of the high ΔΨ because the intracellular negative ion capacities of alkaliphiles are much higher than those of neutralophiles. There are several variations in the adaptation to alkaline environments by bacteria. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiration. To explain the efficient ATP production occurring in H+-less and air-limited environments in alkaliphilic bacteria, we propose a cytochrome c-associated “H+ capacitor mechanism” as an alkaline adaptation strategy.

As an outer surface protein, cytochrome c-550 from Bacillus clarkii possesses an extra Asn-rich segment between the region anchored to the membrane and the main body of the cytochrome c. This structure may contribute to the formation of the proton-binding network to transfer H+ at the outer surface membrane in obligate alkaliphiles. The H+ capacitor mechanism is further enhanced under low-aeration conditions in both alkaliphilic Bacillus spp. and the Gram-negative alkaliphile Pseudomonas alcaliphila.

Bacteria that thrive under extreme environmental conditions, such as low or high temperatures and high or low pH, are called extremophiles. Each extremophile possesses certain strategies for adaptation under different conditions. These complex strategies consist of environmental adaptation mechanisms, such as alterations in specific characteristics of enzymes and membranes. Although there is a common fundamental basis for extremophile metabolism, these organisms differ slightly in the characteristics of certain components necessary for environmental adaptation (e.g., modification of cell surface structures and protein characteristics and production of molecular chaperones).

Alkaliphiles are defined as microorganisms that exhibit better growth at pH ≥ 9 than at pH < 9. Since Vedder (1934) isolated the obligate alkaliphile Bacillus alcalophilus, many alkaliphilic Bacillus strains have been isolated from common environments such as garden soil and horse manure in the search for enzymatic resources, as alkaliphilic Bacillus strains produce heat-stable enzymes (Horikoshi and Akiba, 1982; Horikoshi and Grant, 1998; Horikoshi, 2006, 2011). Although various strains have been isolated, until Nielsen et al. (1995) proposed nine new Bacillus spp., it was not known whether alkaliphilic bacteria comprise multiple species due to the lack of a molecular identification method concomitant with a gene sequence database. Since the discovery made by Nielsen et al. (1995) many additional alkaliphilic bacteria have been isolated and identified as new species. Related reports indicate that diverse alkaliphiles are distributed in a variety of environments, which may indicate the presence of numerous small alkaline niches [e.g., the termite gut (Thongaram et al., 2003)] and/or large high-pH environments (e.g., alkaline lakes). Alkaline environments have been present throughout Earth’s history. Zavarzin (1993) hypothesized that modern soda lakes may represent a refuge for relict terrestrial communities from ancient continents of the Early Proterozoic Eon. In contrast, a leading hypothesis suggests that the origin of life can be track back to ocean-floor-based alkaline hydrothermal vents (Lane and Martin, 2012). Natural H+ gradients across the membranes of iron monosulfide bubbles could lead to the formation of protocells (Russell and Hall, 1997).

The phylogenetic diversity and wide distribution of alkaliphiles on Earth indicates that the evolution of alkaliphiles is not a specific phenomenon but a common event in natural environments. Therefore, many variations in alkaline adaptation mechanisms should be present in alkaliphiles. The range of reported alkaline adaptation mechanisms has not been explained to date. However, diverse adaptation mechanisms involving secondary cell wall variations were reported by Aono and Horikoshi (1983), Aono et al. (1993, 1995,1999). The formation of a negatively charged secondary cell wall results in the pH at the outer surface membrane being lower than the extracellular pH (i.e., medium pH) (Tsujii, 2002). The diversity of environmental adaptation mechanisms underlying the survival of numerous alkaliphiles of different taxa provides various possible explanations for the existence of life in alkaline environments. Alkaliphilic Bacillus spp. have an acidic secondary cell wall. B. halodurans C-125 produces an acidic secondary cell walls consisting of teichurono-peptide and teichuronic acid. In contrast, B. pseudofirmus OF4 cells produces the cell surface protein SlpA and polyglutamic acid (Gilmore et al., 2000). These structures attract H+ and repel OH-, and the structural functions of those components protects the intracellular metabolic pathways from severe extracellular environments. Although the acidic secondary cell wall is indispensable for alkaline adaptation in alkaliphilic Bacillus spp., the variations in the secondary cell wall structure, which are shared among alkaliphilic Bacillus spp., have not been elucidated to date.

Alkaline environments are not always favorable for alkaliphilic bacteria. Alkaliphilic Bacillus spp. have been reported to produce acid to reduce the pH when the ambient pH is too high for metabolism (Horikoshi, 2006). This acid production can often be observed even in media lacking sugars. In contrast, these bacteria create an alkaline environment when the ambient pH is too low for metabolism. These phenomena indicate that several alkaliphiles have the ability to increase the favorability of the ambient environment. Amphibacillus iburiensis is able to grow in broth medium adjusted to pH 11 prior to inoculation (Hirota et al., 2013). However, this bacterium exhibits distinct growth initiation at pH 9 after lowering the pH of the medium via acid production, as observed by monitoring the change in pH during incubation.

Alkaliphiles adapt to the environment by employing various combinations of mechanisms to adjust to alkaline conditions. Alkaliphilic Bacillus strains have been reported to adjust the intracellular pH to an appropriate level via the Na+/H+ antiporter, Na+ channels or stator force generator that drives Na+-dependent motility (Kitada et al., 1982; Ito et al., 2004a,b; Padan et al., 2005), which contribute to replace the H+-potential base transport system with the Na+-potential base transport system. Thus, the Na+/H+ antiporter and other Na+ or K+-related transport systems reduce the utilization of H+ by transport systems, which is very important for alkaliphiles that thrive at one-thousandth the concentration of H+ found under neutral conditions. In addition, the rotational torque of the flagella of alkaliphilic Bacillus strains is produced by the influx of Na+ derived from the Na+-base potential across the membrane (Ito et al., 2004a).

It is reasonable to consider that the entire solute transport system functions via the Na+ potential across the membrane. However, the ATP synthase-based energy production system is derived from the H+-base potential across the membrane (Dimroth and Cook, 2004), which may indicate localization of the H+-base potential across the membrane in the vicinity of the respiratory chain in the horizontal direction. In addition, if H+ is not attracted to the interface in the vicinity of the outer surface membrane, the H+ concentration will be quite low. The H+-base potential may also be present in the vicinity of the outer surface membrane in the perpendicular direction.

Alkaliphilic Bacillus spp. have been reported to exhibit higher membrane electrical potentials (ΔΨ) than neutralophilic Bacillus strains (Yumoto, 2003; Goto et al., 2005). However, the calculation of bulk-base parameters [ΔΨ and transmembrane pH gradient (ΔpH)] for the Δp driving F1Fo-ATPase does not account for ATP production because the high deficiency in ΔpH is not compensated by ΔΨ. We demonstrated the importance of a large ΔΨ in alkaliphiles by showing the contribution of ΔΨ to the retention of H+ in the vicinity of the outer surface of the membrane in the vertical direction and the contribution of efficient ATP production under conditions involving H+ scarcity (Yoshimune et al., 2010). Furthermore, ΔΨ ensures efficient ATP production to enhance the F1Fo-ATPase driving force per H+. In addition, structural and physicochemical mechanisms to retain H+ at the outer surface of the membrane are indispensable. However, it could be difficult to utilize high ΔΨ in the low aeration condition due to the low activity of respiratory system. In this context, we highlight “a high-potential H+ capacitor mechanism” based on the existence of membrane-bound or periplasmic cytochrome c in alkaliphiles.

According to Peter Mitchell’s chemiosmotic theory (Mitchell, 1961), the Δp across the membrane that drives F1Fo-ATPase to produce ATP consists of ΔpH (intracellular pH minus extracellular pH) and the difference in electrical potential across the membrane, i.e., ΔΨ (intracellulary electronegative and extracellularly electropositive across the membrane). The Δp can be calculated by the following formula.

R = gas constant (8.315 J mol-1); T = absolute temperature (298 K = 25°C); F = Faraday constant (96.485 kj [v⋅mol]-1).

The H+ gradient is also utilized for other energy-requiring processes such as transmembrane solute transport and signaling. In general, the parameters Δp, ΔΨ, and ΔpH apply to the bulk-base. Although it is difficult to estimate the values of ΔΨ and ΔpH based on the vicinity of the membrane surface, localization of these parameters in the vicinity of the membrane surface in both the horizontal and vertical directions should be considered, especially in the case of the outer surface membrane of alkaliphilic Bacillus spp. (Gennis, 2016; Sjöholm et al., 2017). These characteristics are common to biological energy production mechanisms. Alkaliphilic bacteria must possess distinctive characteristics at the outer surfaces of the membrane to effectively utilize existing stores of H+ at the outer surface membranes.

Although bulk-base calculations cannot account for ATP production by alkaliphilic Bacillus spp. Due to the great hindrance conferred by negative ΔpH values, the growth of these bacteria is vigorous under alkaline conditions (Guffanti and Hicks, 1991). The growth features of facultative alkaliphilic B. pseudofirmus OF4 were estimated at a steady state under various pH-controlled culture conditions. Strain OF4 exhibits specific growth rates of 0.77 and 1.10 h-1 in media maintained at pH values of 7.5 and 10.6, respectively. We estimated the growth characteristics of the obligate alkaliphilic B. clarkii K24-1U in batch culture. Strain K24-1U exhibited more rapid growth (μmax = 0.33 h-1) than the neutralophilic B. subtilis IAM 1026 (μmax = 0.26 h-1) (Hijikata, 2004). These superior growth characteristics are attributable to vigorous ATP production. Thus, in alkaliphilic Bacillus spp., these growth characteristics are not accounted for by bulk-based bioenergetic parameters.

The zone formed in the vicinity of the outer surface of the membrane formed due to the presence of phosphate and carbonyl headgroups stabilizes excess hydrated H+ relative to the bulk solution. However, the pH of the bulk phase at the outer side membrane in alkaliphiles is expected to be at least 2 units higher than that in neutralophiles. In addition, the rate of H+ exchange between the deep interface zone in the vicinity of the outer surface membrane and the bulk phase in alkaliphiles may be greater than that in neutralophiles due to the large difference in pH between the deep interface zone and the bulk phase at the outer membrane (Mulkidjanian et al., 2006; Gennis, 2016). Therefore, it survival under proton-less conditions without specific alkaline adaptation mechanisms is difficult. Although many neutralophilic Bacillus spp. can grow at pH 9, most cannot grow well at pH 10, unlike alkaliphilic Bacillus spp.

The H+ transfer mechanism on the outer surface of the membrane is dependent on the distance between the H+-vent (e.g., a respiratory complex such as cytochrome c oxidase) and H+-sink (e.g., F1Fo-ATPase). Sjöholm et al. (2017) investigated the distance effect between the terminal oxidase, cytochrome bo3 and F1Fo-ATPase by reconstituting these proteins in a vesicle and measuring ATP synthesis activity. An even shorter distance between the H+-vent and H+-sink than that observed in neutralophiles was expected to be favorable in alkaliphiles. A high cytochrome content has been reported in alkaliphilic Bacillus spp. (Lewis et al., 1980; Guffanti et al., 1986; Yumoto et al., 1997; Goto et al., 2005). This feature will relate to H+ behavior at the outer surface membrane.

It is considered that the H+ concentration at the outer surface membrane in alkaliphiles is lower than that in neutralophiles due to the low background H+ concentration in alkaliphiles. In addition, the rate constant of H+ exchange between the deep interface zone of the outer surface membrane and the adjacent bulk zone may be higher in alkaliphiles than in neutralophiles due to the larger pH gap between the deep interface zone of the outer surface membrane and the adjacent bulk zone in alkaliphiles than that in neutralophiles. In addition to the membrane-surface-based ΔpH (ΔpH values) (Xiong et al., 2010), membrane-surface-based ΔΨ values may also exist (Lyu and Lazár, 2017). In addition, proteins present on the outer surface membrane may play a role in the retention of H+ in this region. A comparative experiment to detect H+ in the bulk phase during respiration in the obligate alkaliphilic B. clarkii K24-1U and the neutralophilic B. subtilis IAM 1026 was performed by monitoring the change in pH (Yoshimune et al., 2010). Whole-cell suspensions of both Bacillus stains consumed oxygen immediately after the introduction of oxygen, and a lag period was observed for H+ extrusion by the respiratory chain into the bulk phase. The lag period for alkaliphilic B. clarkii K24-1U was significantly longer at pH 10 than at other pH values and significantly longer than those for B. subtilis at various pH values. This observation may indicate that H+ was transferred into the bulk phase after all the H+ retention sites were occupied by H+ translocated via respiratory complexes.

The introduction of monensin, which exchanges extracellular H+ for intracellular Na+, similar to a Na+/H+ antiporter, resulted in a prolonged lag period for H+ extrusion to the bulk, indicating that the H+ present at H+ retention sites on the outer surface membrane translocated to the intracellular side via countertransport with the Na+ present in the intracellular space. In contrast, when a ΔΨ-disrupting agent such as valinomycin or ETH-157 was introduced, the lag phase disappeared. These experiments reveal the meaning of ΔΨ values in alkaliphilic Bacillus spp. are larger than those in neutralophilic Bacillus spp.

The growth of alkaliphilic Bacillus spp. is much faster than that of neutralophilic Bacillus spp. as described above, which may be attributable to the higher ATP production rate in alkaliphilic Bacillus spp. than that in neutralophilic Bacillus spp. The ATP production rate was estimated using the obligate alkaliphile B. clarkii DSM 8720T and neutralophilic B. subtilis IAM 1026 (Hirabayashi et al., 2012). B. clarkii DSM 8720T produced 7.2 nmol ATP⋅mg protein-1⋅min-1 (endogenous substrate) at pH 10, which was comparable to the amount produced by B. pseudofirmus OF4 (6.6 ± 3.9 nmol ATP⋅mg protein-1⋅min-1 [starved cells re-energized with malate]) at pH 10.5 (Guffanti and Krulwich, 1992). In contrast, B. subtilis IAM 1026 produced 0.96 nmol ATP⋅mg protein-1⋅min-1 at pH 7. Thus, the ATP production rate in alkaliphilic Bacillus spp. was much higher (6.9-7.5 times) than that in neutralophilic B. subtilis.

If rapid ATP production by alkaliphilic Bacillus spp. was attributable to rapid H+ translocation across the membrane by respiratory complexes, then these bacteria would exhibit a rapid O2 consumption and/or high efficiency of H+ translocation across the membrane per molecule of O2 consumed. However, the oxygen consumption rate of B. clarkii DSM 8720T cells was 0.19 μmol O2⋅min-1⋅mg cell protein-1 at pH 10. Although these data were obtained with an endogenous substrate, the results were comparable to those obtained using malate as the substrate in other alkaliphilic Bacillus spp. (Lewis et al., 1980; Guffanti et al., 1986; Aono et al., 1996). The oxygen consumption rate of B. subtilis IAM 1026 was 0.50 μmol O2⋅min-1⋅mg cell protein-1 at pH 7. Thus, the oxygen consumption rate of whole B. subtilis IAM 1026 cells at pH 7 was 2.6 times higher than that of B. clarkii DSM 8720T at pH 10. Unlike the theoretical H+/O ratio calculated for Bacillus spp. (complex III, 4 plus complex IV, 2 = 6) (Sone et al., 1999), the H+/O ratio in B. clarkii DSM 8720T was 3.6 at pH 10. In contrast, the H+/O ratio in B. subtilis IAM 1026 was 5.3. The H+/O ratio in B. subtilis IAM 1026 was 1.5 times higher than that in B. clarkii DSM 8720T (Goto et al., 2016). Considering both the O2 consumption rate and the H+/O ratio in both strains, the H+ translocation rate in B. subtilis IAM 1026 (pH 7) was 3.9 times higher than that in B. clarkii DSM 8720T (pH 10). This finding indicated that the higher ATP production rate in neutralophilic B. subtilis IAM 1026 than that in alkaliphilic B. clarkii DSM 8720T cannot be accounted for by the rate of H+ translocation across the membrane via the respiratory chain. The large differences in ATP production rates between B. clarkii DSM 8720T and B.

subtilis IAM 1026 are attributable to differences in the F1Fo-ATPase driving force per H+, which may arise from differences in ΔΨ. It is difficult to translocate H+ from the intracellular to the extracellular side of the membrane because ΔΨ hinders translocation in that direction by electrical attraction. However, once translocated to the outer surface of the membrane, H+ has high potential to drive F1Fo-ATPase. It has been reported that the ΔΨ in alkaliphilic Bacillus spp. is larger than that in neutralophilic Bacillus spp. (Hoffmann and Dimroth, 1991; Krulwich, 1995; Yumoto, 2002; Goto et al., 2005; Hirabayashi et al., 2012). The same phenomenon has been observed in the alkaliphilic B. clarkii DSM 8720T and neutralophilic B. subtilis IAM 1026. The ΔΨ values of B. clarkii DSM 8720T and B. subtilis IAM 1026 were -192 mV at pH 10 and -122 mV at pH 7, respectively (Goto et al., 2016).

As described above, ΔΨ is very important for attracting H+, which translocates via the respiratory chain to the outer surface membrane. Generally, the ΔΨ across the membrane is generated by translocation of positively charged H+ from the intracellular to the extracellular region across the membrane. However, the larger ΔΨ in alkaliphilic Bacillus spp. than that in neutralophilic Bacillus spp. cannot be accounted for by the rate of H+ translocation. The large ΔΨ values of alkaliphilic Bacillus spp. may be attributed to the production of high Donnan potentials (Donnan, 1924). The Donnan effect is observed in the presence of membrane-impermeable charged molecules on only one side of the membrane. If a membrane-impermeable negatively charged molecules are present only on the intracellular side of the membrane, these molecules contribute to the formation of the ΔΨ. To estimate the contribution of the Donnan effect to the large ΔΨ in alkaliphilic Bacillus spp., intracellular negative ion capacity was estimated in the obligate alkaliphilic B. clarkii DSM 8720T and facultative alkaliphilic B. cohnii YN-2000 and compared to that of the neutralophilic B. subtilis IAM 1026. To estimate the intracellular negative ion capacity, a cell extract containing inside-out membrane vesicles was prepared and titrated using the positively charged substance clupein sulfate. Negative ion capacity is the amount of negative surface charge of substances in a solution (substances in the intracellular fraction of bacterial cells in this study) estimated by the colloid titration method. This method is one procedure by which the net charge density of surfaces, polyelectrolytes levels, and charge demand of colloidal materials in a solution may be estimated. The measured parameter is the capacity of the mixture to adsorb a polyelectrolyte with the opposite net charge.

The intracellular negative ion capacity in alkaliphilic Bacillus spp. increased with increasing pH in the range of pH 6-8, whereas the intracellular negative ion capacity of neutralophilic B. subtilis IAM 1026 changed very little within this pH range. To understand the corresponding negative ion capacity when alkaliphilic Bacillus spp. were grown under alkaline conditions, the intracellular pH of the alkaliphilic strains was estimated at an extracellular pH of 10 (Goto et al., 2016). The intercellular pH of the alkaliphilic Bacillus spp. B. clarkii DSM 8720T and B. cohnii YN-2000 was 8.1, which is comparable to values reported previously using other alkaliphilic Bacillus spp. (Guffanti et al., 1986; Guffanti and Hicks, 1991). At this intracellular pH, the negative ion capacities of B. clarkii DSM 8720T and B. cohnii YN-2000 were 2.9 and 3.3 (×106 eq⋅mg protein-1), respectively. The intracellular pH of neutralophilic B. subtilis IAM 1026 was 6.7 when the extracellular pH was 7. At this intracellular pH, the negative ion capacity of B. subtilis IAM 1026 was 0.7 (×106 eqmg protein-1). This finding indicates that alkaliphilic Bacillus spp. possess a much higher intracellular negative ion capacity than neutralophilic B. subtilis. This high intracellular negative ion capacity contributes to the intrinsic ΔΨ. There may be questions regarding the existence of such a high intracellular negative ion capacity. One explanation is that the intracellularly expressed acidic proteins in alkaliphilic Bacillus spp. are negatively charged at slightly higher intercellular pH values (ca. pH 8) than those in neutralophiles (pH 6-7). Whole-genome analyses have been performed previously in alkaliphilic Bacillus spp. as well as neutralophilic Bacillus spp., and Janto et al.

(2011) estimated the average pI values of proteins localized in intracellular and extracellular spaces, the cell wall and the membrane. Despite a tendency toward a high frequency of acidic proteins in the cell wall and extracellular proteins in alkaliphiles, the obligate alkaliphile B. selenitireducens ML10 possesses much higher levels of low pI proteins than neutralophiles. The combination of the acidic nature of the intracellular side chains of acidic membrane proteins and intercellular acidic proteins is predicted to contribute to the high negative ion capacity in alkaliphilic Bacillus spp.

The primary role of cytochrome c is to transfer electrons between complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase) proteins. A typical class I cytochrome c possesses one low-spin heme c in the N-terminal region bound to the protein by two thioether bonds with cysteine residues (Ambler, 1991). The proximal side and distal side of the iron ligands have a histidine residue and a methionine residue, respectively. The molecular weight of cytochrome c is approximately 8,000-14,000, and cytochrome c is a soluble protein in mitochondria and Gram-negative bacteria (Pettigrew and Moore, 1987; Moore and Pettigrew, 1990; Yamanaka, 1992; Brayer and Murphy, 1996).

Although numerous studies have assessed soluble cytochromes c from various sources, including mitochondria and Gram-negative bacteria, there have been limited examples of cytochrome c from Gram-positive bacteria. Despite having some outer surface membrane space, Gram-positive bacteria do not have outer membranes similar to those of Gram-negative bacteria. Therefore, Gram-positive bacteria do not possess a periplasmic space equivalent to that in Gram-negative bacteria. Consequently, all the cytochrome c in Gram-positive bacteria is membrane-bound. For example, B. subtilis possesses two types of membrane-bound cytochromes: c-550 and c-551. Cytochrome c-550 has a molecular mass of 13 kDa, with a membrane-anchored domain consisting of a single α-helical transmembrane segment of a hydrophobic polypeptide containing 30 amino acids (von Wachenfeldt and Hederstedt, 1990). The midpoint redox potential of cytochrome c-550 is +178 mV (von Wachenfeldt and Hederstedt, 1993). The other cytochrome c, namely, c-551, has a molecular mass of 10 kDa and binds to the membrane via a diacyl-glyceryl-cysteine moiety (Bengtsson et al., 1999). The midpoint redox potential of cytochrome c-551 is >100 mV. The functions of these two membrane-binding cytochromes c have not been characterized. However, it is expected that these proteins are involved in transport of electrons between complexes III and IV.

It has been reported that alkaliphilic Bacillus spp. contain higher amounts of membrane-bound cytochrome c than neutralophilic B. subtilis (Yumoto et al., 1997; Hijikata, 2004). In addition, the amount of membrane-bound cytochrome c is low in mutant strains that lack the ability to grow in alkaline media (Lewis et al., 1980). These findings suggest that membrane-bound cytochrome c in alkaliphiles may contribute to adaptation in alkaline environments. Cytochrome c-552 from B. pseudofirmus RAB was first purified and characterized from alkaliphilic Bacillus spp., exhibiting a molecular mass of 16.5 kDa and midpoint redox potential of +66 mV at pH 7, which decreases as the surrounding pH is increased (Davidson et al., 1988). This cytochrome c is normally membrane bound but was purified as a soluble protein. Cytochrome c proteins are predicted to play a role in electron transport between complexes III and IV.

The primary and 3D structures of cytochrome c were first studied in alkaliphilic Bacillus-related taxa using cytochrome c-553 purified from Sporosarcina pasteurii (formerly B. pasteurii) (Benini et al., 2000). Cytochrome c-553 has a low molecular mass of 9.6 kDa and a low midpoint redox potential (+47 mV) (Benini et al., 1998). The crystal structure of the protein exhibits a highly asymmetric charge distribution. Most of the charges are located on the side opposite to that exposed to the heme edge. The localization of charges is related to H+ transfer on the outer surface membrane. Analyses of the physicochemical parameters reveal that the heme solvent accessibility is correlated with entropy. This finding suggests a direct link between the major determinant of the electrochemical potential (entropy) and a structural parameter (heme solvent exposure). The low midpoint redox potential of cytochrome c-553 could be explained by the decrease in reduction entropy via extrusion of water molecules from the protein hydration shell, affecting a large number of water molecules in the case of increased solvent accessibility, which occurs upon heme reduction.

The abundance of alkaliphilic Bacillus spp.-specific membrane-bound cytochrome c (Yumoto et al., 1991) has also been studied using the facultative alkaliphile B. cohnii YN-2000 (Yumoto et al., 2000). The abundance of membrane-bound cytochrome c is higher during growth at pH 10 than during growth at pH 7. In addition, cytochrome abundance is further increased under low-aeration conditions. Solubilized B. cohnii YN-2000 membranes prepared from cells grown at pH 10 using the detergent Triton X-100 contain larger amounts of cytochrome c-553 than solubilized membranes prepared with cells grown at pH 7. The native molecular mass of cytochrome c-553, which was solubilized by Triton X-100 in a buffer, was determined to be 127 kDa by gel filtration. The molecular mass of Triton X-100, which was used to solubilize cytochrome c-553 in solution is 90 kDa. Therefore, if cytochrome c-553 is contained in micelles of Triton X-100, the actual native molecular mass should be 37 kDa. The stoichiometry of cytochrome c-553 and Triton X-100 is considered to be 1:1. In contrast, the molecular mass of cytochrome c-553 was determined to be 10,500 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results described above suggest that cytochrome c-553 forms a tetramer in its native form in solution or in the original membrane. Cytochrome c-553 from B. cohnii YN-2000 also exhibits a low midpoint redox potential of +87 mV at the pH values ranging from 6 to 8.

A novel type of cytochrome c oxidase, cytochrome aco3, was purified and characterized from B. cohnii YN-2000 (Qureshi et al., 1990; Yumoto et al., 1993; Denda et al., 2001). Cytochrome c-533 can react with cytochrome aco3, and the reaction is greatly enhanced in the presence of the positively charged substance poly-L-lysine, which may accelerate binding to two negatively charged molecules: cytochrome c-553 and cytochrome aco3. Although cytochrome aco3 was present in equal amounts in cells grown at pH 10 and pH 7, the cytochrome c-553 content was higher in cells grown at pH 10 than in those grown at pH 7. The midpoint redox potential of the attached cytochrome c was +95 mV at pH 7 (Orii et al., 1991). In contrast, cytochrome a exhibited two forms with midpoint redox potentials of +250 mV and +323 mV at pH 7 (Orii et al., 1991). A stopped-flow study on cytochrome aco3 showed that the cytochrome a component exhibited the highest affinity for electrons and only a minimal contribution to O2 reduction among the involved redox components. Therefore, membrane-bound cytochrome c-553 may directly react not only with cytochrome c in cytochrome aco3 but also with the cytochrome a moiety in cytochrome aco3. During electron flow from cytochrome c-553 or cytochrome c to cytochrome a in cytochrome aco3, there is a large midpoint redox potential difference between each component. This significant difference is necessary for electron flow between each cytochrome c and cytochrome a in cytochrome aco3 to overcome the large ΔΨ (Yumoto et al., 1993). Therefore, the large difference in redox potential between the outer surface redox components and intramembrane redox components sustains electron transfer in the respiratory system in alkaliphilic Bacillus spp.

In summary, this large difference in midpoint redox potential may be necessary for the generation of produce the large energy potential required for the translocation of intracellular H+ to overcome the hindrance conferred by the high ΔΨ and to retain H+ on the intracellular side of the membrane.