Biogenesis of cbb(3)-type cytochrome c oxidase in Rhodobacter capsulatus

Abstract

The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb(3)-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b(3)-Cu(B) center, have to be coordinated precisely both temporally and spatially to yield a functional cbb(3)-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb(3)-Cox, and provide a highly tentative model for cbb(3)-Cox assembly and formation of its heme b(3)-Cu(B) binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Figure 1

The respiratory chain of R. capsulatus and its regulation in response to oxygen availability. The protein complexes and electron carrier of the respiratory chain are shown in yellow. UQ and UQH₂ correspond to the oxidized and reduced form of quinones. Soluble cytochrome c₂ and the membrane-bound cytochrome c_y (indicated as c in the figure) are electron donors for the photosynthetic reaction center and cbb₃-Cox. Metabolic processes which also consume redox equivalents are shown in blue. Regulatory proteins are shown in grey. RegB is a sensor kinase that phosphorylates the response regulator RegA under low oxygen concentrations. Phosphorylated RegA (RegA-P) inhibits (shown as the red square) expression of ccoNOQP (cbb₃-Cox) and hupSLC ([NiFe] hydrogenase), but stimulates the expression of the bd-type quinol oxidase and of genes required for photosynthesis. In addition, RegA-P stimulates (shown as an arrow) the expression of nifHDK (nitrogenase) and the cbb₁ and cbb₂ operons (CO₂ fixation) both directly and indirectly [154]. Under microaerobic and anaerobic conditions HvrA further represses ccoNOQP expression but stimulates bd-type quinol oxidase expression. Finally, FnrL probably activates ccoNOQP expression in the transition phase between aerobic and anaerobic conditions. R. capsulatus SenC and its R. sphaeroides homologue PrrC were suggested to be involved in oxygen-dependent expression of ccoNOQP and photosynthetic genes. FixLJ-K regulatory system discussed in the text is not shown in the figure.

Figure 2

A. 3D structures of different types of heme-Cu:O₂ reductases and NO reductase: Catalytic subunit I blue (cbb₃-Cox CcoN), subunit II (cbb₃-Cox CcoO) – magenta, subunit III (cbb₃-Cox CcoP) – green, additional subunit –orange. B. Architecture of the catalytic binuclear center of the different types of heme-Cu:O₂ reductases and NO reductase. Heme – red, His – green, Tyr – violet, Glu – orange. Spheres: copper – blue, heme iron - dark grey, non-heme iron – light grey, calcium – yellow. The structures depicted are taken from protein database (PDB) entries PDB ID: 3MK7 (cbb₃-Cox) [14], PDB ID: 3O0R (NOR ) [60], PDB ID: 1M56 (aa₃-Cox) [19], PDB ID: 1EHK (ba₃-Cox) [17] using Jmol software (http://www.jmol.org/).

Figure 3

A. An assembly scheme for cbb₃-Cox in R. capsulatus. Using BN-PAGE, active cbb₃-Cox is detectable in R. capsulatus membranes as a 230 kDa complex. Assembly of the 230 kDa complex proceeds via two inactive assembly intermediates: a 210 kDa subcomplex that contains the catalytic subunit CcoN, the mono-heme cytochrome c subunit CcoO and the single-spanning membrane protein CcoH, and a 40 kDa subcomplex composed of the di-heme cytochrome c subunit CcoP, the small subunit CcoQ and a second copy of CcoH. Full assembly of cbb₃-Cox is probably achieved by the dimerization of the cytoplasmic domains of the two CcoH. CcoH was originally considered to function as an assembly factor, but recent data demonstrate that it is a stable component of the fully assembled cbb₃-Cox. The c-type cytochromes in both assembly subcomplexes have their heme groups attached indicating that c-type cytochrome maturation preceded the formation of the assembly intermediates. B. A tentative model for the maturation of CcoN subunit. The maturation of CcoN requires Cu and heme b insertions. Cu is probably imported into the cytoplasm of R. capsulatus by the major facilitator superfamily (MFS) protein CcoA as oxidized Cu²⁺. On the cytoplasmic face of the membrane Cu²⁺ is reduced to Cu⁺ by the ferredoxin-like protein CcoG and transported back to the periplasmic face of the membrane by the PIB-type ATPase CcoI. CcoI then supplies Cu⁺ to CcoN either directly within the membrane or from the periplasm, or via some putative periplasmic Cu binding proteins such as SenC and PCuAC. The insertion of heme b and heme b₃ into CcoN is probably mediated by the assembly factor CcoS.

Figure 4

Cytochrome c maturation (Ccm) system in Rhodobacter capsulatus. The cytochrome c maturation components can be divided into three modules. First module (right) is the transport of heme and its preparation for ligation to apocytochrome that involves CcmABCD proteins which forms a ABC-type transporter that has a role in the delivery of heme to the heme chaperone CcmE before the ligation. It is unknown whether heme is transported via CcmABCD complex or by another unknown protein. CcmA and CcmB are required for the release of heme-bound CcmE from CcmC and CcmD. CcmC and CcmD are involved in the heme attachment to CcmE. Second module (left) involves the apocytochrome thioredox and chaperoning processes. After the translocation of apocytochrome into the periplasm, the cysteine thiols are first oxidized by DsbA-DsbB, then reduced by CcdA, CcmG and/or CcmH. Third module (middle) consists of CcmHIF which is the heme ligation core complex. CcmI binds to C-terminal part of apocytochrome to deliver it to the core complex for the catalysis of the thioether bond formation between reduced apocytochrome and heme vinyl groups forming the mature cytochrome c (adapted from [155])