Fritz G, Griesshaber D, Seth O, Kroneck P M
Fachbereich Biologie, Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, 78457 Konstanz, Germany.
Biochemistry. 2001 Feb 6;40(5):1317-24. doi: 10.1021/bi001480+.
A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.
从硫酸盐还原菌脱硫脱硫弧菌埃塞克斯菌株的可溶性组分和膜组分中纯化出一种九聚体细胞色素c至同质。对编码该蛋白质的基因进行了克隆和测序。这种多聚体细胞色素c的一级结构与脱硫脱硫弧菌ATCC 27774的九聚体细胞色素c以及普通脱硫弧菌希登伯勒菌株的16聚体HmcA蛋白的一级结构高度同源。对脱硫脱硫弧菌埃塞克斯菌株中编码九聚体细胞色素c的基因下游序列的分析揭示了一个编码HmcB同源物的开放阅读框。这种操纵子结构表明脱硫脱硫弧菌埃塞克斯菌株中存在Hmc复合物，其中九聚体细胞色素c取代了普通脱硫弧菌中发现的16聚体HmcA蛋白。分析了脱硫脱硫弧菌埃塞克斯菌株的九聚体细胞色素c在氧化态和还原态下的分子和光谱参数。还原后，当从Fe(III)态转变为Fe(II)态时，该蛋白质的pI从8.25显著变为5.0。迄今为止，细胞色素中尚未报道过这种由氧化还原诱导的pI变化；这很可能是蛋白质结构构象重排的结果，圆二色光谱证实了这一点。将九聚体细胞色素c对[Ni,Fe]氢化酶的反应性与四聚体细胞色素c(3)的反应性进行了比较；细胞色素c(3)和周质[Ni,Fe]氢化酶均源自脱硫脱硫弧菌埃塞克斯菌株。九聚体蛋白对[Ni,Fe]氢化酶表现出亲和力和反应性[K(M)=20.5±0.9 microM；v(max)=660±20 nmol还原型细胞色素min(-1)(nmol氢化酶)(-1)]，与细胞色素c(3)[K(M)=12.6±0.7 microM；v(max)=790±30 nmol还原型细胞色素min(-1)(nmol氢化酶)(-1)]相似。这表明九聚体细胞色素c是[Ni,Fe]氢化酶的一种有效的生理电子受体。
