Minimal Influence of [NiFe] Hydrogenase on Hydrogen Isotope Fractionation in H2-Oxidizing Cupriavidus necator

Brian J Campbell; Alex L Sessions; Daniel N Fox; Blair G Paul; Qianhui Qin; Matthias Y Kellermann; David L Valentine

doi:10.3389/fmicb.2017.01886

Minimal Influence of [NiFe] Hydrogenase on Hydrogen Isotope Fractionation in H₂-Oxidizing Cupriavidus necator

Front Microbiol. 2017 Oct 4:8:1886. doi: 10.3389/fmicb.2017.01886. eCollection 2017.

Authors

Brian J Campbell¹, Alex L Sessions², Daniel N Fox³, Blair G Paul⁴, Qianhui Qin⁵, Matthias Y Kellermann⁶, David L Valentine^{1

4}

Affiliations

¹ Department of Earth Science, University of California, Santa Barbara, Santa Barbara, CA, United States.
² Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, United States.
³ Undergraduate College of Letters and Sciences, University of California, Santa Barbara, Santa Barbara, CA, United States.
⁴ Marine Science Institute, University of California, Santa Barbara, Santa Barbara, CA, United States.
⁵ Interdepartmental Graduate Program in Marine Science, University of California, Santa Barbara, Santa Barbara, CA, United States.
⁶ Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University, Wilhelmshaven, Germany.

Abstract

Fatty acids produced by H₂-metabolizing bacteria are sometimes observed to be more D-depleted than those of photoautotrophic organisms, a trait that has been suggested as diagnostic for chemoautotrophic bacteria. The biochemical reasons for such a depletion are not known, but are often assumed to involve the strong D-depletion of H₂. Here, we cultivated the bacterium Cupriavidus necator H16 (formerly Ralstonia eutropha H16) under aerobic, H₂-consuming, chemoautotrophic conditions and measured the isotopic compositions of its fatty acids. In parallel with the wild type, two mutants of this strain, each lacking one of two key hydrogenase enzymes, were also grown and measured. In all three strains, fractionations between fatty acids and water ranged from -173‰ to -235‰, and averaged -217‰, -196‰, and -226‰, respectively, for the wild type, SH^- mutant, and MBH^- mutant. There was a modest increase in δD as a result of loss of the soluble hydrogenase enzyme. Fractionation curves for all three strains were constructed by growing parallel cultures in waters with δD_water values of approximately -25‰, 520‰, and 1100‰. These curves indicate that at least 90% of the hydrogen in fatty acids is derived from water, not H₂. Published details of the biochemistry of the soluble and membrane-bound hydrogenases confirm that these enzymes transfer electrons rather than intact hydride (H^-) ions, providing no direct mechanism to connect the isotopic composition of H₂ to that of lipids. Multiple lines of evidence thus agree that in this organism, and presumably others like it, environmental H₂ plays little or no direct role in controlling lipid δD values. The observed fractionations must instead result from isotope effects in the reduction of NAD(P)H by reductases with flavin prosthetic groups, which transfer two electrons and acquire H⁺ (or D⁺) from solution. Parallels to NADPH reduction in photosynthesis may explain why D/H fractionations in C. necator are nearly identical to those in many photoautotrophic algae and bacteria. We conclude that strong D-depletion is not a diagnostic feature of chemoautotrophy.

Keywords: Cupriavidus necator; D/H; autotrophic metabolism; fatty acid; hydrogen isotope; hydrogenase.