A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential

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A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly22-Asn34, in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C15, C16 and C17 chain lengths and C15 chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys18. Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys18 → Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.