Adams Hannah R, Krewson Callie, Vardanega Jenny E, Fujii Sotaro, Moreno-Chicano Tadeo, Moreno Tadeo, Sambongi Yoshihiro, Svistunenko Dimitri, Paps Jordi, Andrew Colin R, Hough Michael A
School of Biological Sciences , University of Essex , Wivenhoe Park , Colchester , Essex CO4 3SQ , UK . Email:
Department of Chemistry and Biochemistry , Eastern Oregon University , La Grande , Oregon 97850 , USA . Email:
Chem Sci. 2019 Jan 21;10(10):3031-3041. doi: 10.1039/c8sc05210g. eCollection 2019 Mar 14.
Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome '-β (cyt cp-β). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (NO), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-β (the only known cytochromes ' with a β-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-β may have evolved from cyt P460 a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-β and cyt P460 from the obligate methanotroph, (Bath). These proteins display a similar β-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-β and characterise their phylogeny.
自然界善于利用高度相似的蛋白质折叠来执行截然不同的功能，然而这种功能分化发生的机制仍未得到充分表征。在某些甲烷营养细菌中，已鉴定出两种同源的五配位c型血红素蛋白：细胞色素P460（cyt P460）和细胞色素cp-β（cyt cp-β）。细胞色素P460能够将羟胺转化为一氧化二氮（NO），一种强效温室气体。这种反应性与羟胺氧化还原酶（HAO）相似，HAO是硝化细菌和甲烷营养细菌中的关键酶。Cyt P460和HAO都具有不寻常的蛋白质-血红素交联，由HAO中的一个Tyr残基和cyt P460中的一个Lys形成。相比之下，细胞色素cp-β（唯一已知的具有β-折叠的细胞色素）缺乏这种交联，并且似乎经过优化以结合非极性分子（包括NO和CO）而无需酶促转化。我们的生物信息学分析支持cyt cp-β可能从cyt P460通过基因复制事件进化而来的提议。使用高分辨率X射线晶体学、紫外可见吸收、电子顺磁共振（EPR）和共振拉曼光谱，我们表征了专性甲烷营养菌（巴斯）中cyt cp-β和cyt P460的整体蛋白质折叠和活性位点结构。这些蛋白质显示出相似的β-折叠蛋白质结构，同时血红素口袋区域和局部三级结构发生了变化，将一种羟胺氧化酶转化为一种气体结合蛋白。结构比较为合成酶学的酶重新设计和气体传感器蛋白工程提供了相关见解。我们还展示了细胞色素cp-β的广泛存在并表征了它们的系统发育。
