Figure 7.

Thioreduction of the disulfide bond at the HBS of apocyt c and stereo-specific heme ligation during Ccm. The comprehensive model depicted here uses apocyt c₁ as an example, but the process is considered to be similar with other apocyts c as well. In wild-type cells, after translocation to the periplasm, apocyt c₁ is oxidized by the thiol-disulfide oxidoreductase DsbA, resulting in the formation of a disulfide bond at its HBS (³⁴CXXCH³⁸) (step 1). Ccm-specific thioredoxin CcmG carries a nucleophilic attack via its N-terminal, solvent exposed Cys-75 to the N-terminal Cys-34 at the HBS of apocyt c₁ (steps 2 and 3), forming CcmG^Cys-75–^Cys-34apocyt c₁ mixed disulfide bond between them (step 3). The C-terminal Cys-78 of CcmG resolves this mixed disulfide bond, resulting in reduced apocyt c₁ and oxidized CcmG (step 4), which is then re-reduced by CcdA (via electrons coming from cytoplasmic thioredoxins, not shown) (step 5). Reduced apocyt c₁ attacks oxidized CcmH with its N-terminal Cys-34 (step 6), forming an intermediate in which apocyt c₁ Cys-34 forms a mixed disulfide with the C-terminal highly reactive Cys-45 of CcmH (step 7), defining the stereo-specificity of heme ligation. Once the CcmH^Cys-45–^Cys-34apocyt c₁ intermediate is formed, holoCcmE carrying heme attached via its vinyl-2 is assumed to interact with CcmH and apocyt c₁ (step 8), leading to the formation of the first thioether bond between the available Cys-37 of apocyt c₁ and vinyl-4 of heme. The CcmH–apocyt c₁–heme–CcmE intermediate thus formed is resolved efficiently via Cys-75 of CcmG, attacking this mixed disulfide (step 9), to form a mixed disulfide with Cys-45 of CcmH and releasing apocyt c₁ (step 10). The Cys-34 of apocyt c₁ is then assumed to form the second thioether bond with vinyl-2 of heme and released from CcmE (possibly involving CcmF, not shown). The CcmG^Cys-75–^Cys-45CcmH mixed disulfide bond is resolved via Cys-78 of CcmG, rendering CcmH reduced and CcmG oxidized (step 11). As before (steps 2–4), CcdA recycles oxidized CcmG (step 12), and CcmH is oxidized via DsbA or another periplasmic oxidant(s) (step 13, heavy dashed line). Conceivably, the CcmH^Cys-45–^Cys-34apocyt c₁ intermediate may also be formed by an alternative route via a nucleophilic attack of the HBS disulfide bond by Cys-45 of reduced CcmH (steps 6a–7a), and be resolved via Cys-42 of CcmH, yielding oxidized CcmH (steps 9a–10a). However these steps (indicated by lightly dotted arrows) are less favorable as CcmH is mainly oxidized in vivo, and Cys-42 of CcmH is not very reactive. The thiol–disulfide exchange rates k (10² m⁻¹ s⁻¹, small red arrows) determined in this work are used to develop this scheme, where the resolution of intra-molecular disulfide bonds is indicated in small brown or gray arrows.