Supporting information; Hassan et al. Nitric oxide homeostasis
To estimate turnover rates of c Nor, we need an estimate of the number of protein molecules per cell. Girsch and de Vries (1997) extracted and purified c Nor from anaerobically grown
Paracoccus denitrificans and obtained a nearly pure fraction (on protein-basis) after hydroxyapatite column separation. Based on the measured activity in this fraction and the fact that the activity of c NOR in vitro is approx. 50% of that in intact membranes, 18% of the c Nor was recovered. Based on the protein content in the pure fraction (4.2 mg ) and the protein content of the
4.2
original membrane fraction (2000 mg ), we find that the membrane protein fraction contains 0.18
2000
=
0.0117
g cNor g
-1 membrane proteins , or 167 nmol cNor g
-1
membrane proteins (assuming mol weight of c Nor = 70,000).
Assuming that membrane proteins account for
1
3
to
1
2
of the total protein pools of bacterial cells, we find that the c Nor content per g whole cell protein is 56–84 nmol cNor g -1 cell-protein .
Bergaust et al. (2010, Supplementary Material) measured whole cell protein of Pa. denitrificans to be
96×10 -15 g cell
-1
. With 56–84 nmol cNor g
-1
cell-protein , we find that the amount of c Nor per cell is 5.34–8.02×10
-21
mol cell
-1
= 3212–4818 molecules cell
-1
.
References
Bergaust, L., Mao, Y., Bakken, L.R., and Frostegård, Å. (2010) Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrogen oxide reductase in Paracoccus denitrificans . Appl Environ Microbiol 76 : 6387-6396.
Girsch, P., and de Vries, S. (1997) Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans . Biochim Biophys Acta 1318 : 202-
216.
1

Supporting information; Hassan et al. Nitric oxide homeostasis
Figure S1. Cell-specific rates of 𝐍𝐎 −
𝟐 reduction (𝒗
𝐍𝐎
−
𝟐
) ; results for the anoxic cultures used to produce inoculum for the measurements of NO reduction kinetics. The measured N
2
production
( A ) was used to estimate cell density ( B
) based on previously determined growth yield = 1.79×10 13 cells mol
-1 electrons to NO x
(Bergaust et al., 2010). Panel C shows the NO concentrations ( nM in the liquid ). For each time increment, the cell specific rates of NO −
2
reduction were calculated (Panel
D ) based on the measured rates per vial and the estimated cell numbers. For all the panels, the individual result for the three replicate vials are shown (line = average). The final cell density, measured by OD
660
in the individual vials, was within ±7% of that predicted by cumulative electron flow and the growth yield = 1.79×10 13 cells mol
-1 electrons.
2

Supporting information; Hassan et al. Nitric oxide homeostasis
Figure S2. Recovery of NO-N as N
2
(A) and N
2
O concentrations throughout the NO reduction assay (B). Panel A shows the cumulative NO reduction and N
2
production ( µmol NO and N
2
–N vial
-1
, i.e., in liquid + headspace), closely matching throughout the incubation. Panel B shows the amounts of NO and N
2
O-N ( nmol N vial
-1
) throughout the incubation (logarithmic scale). Both the results are for the same vial as shown in Fig. 4 in the manuscript.
3

Supporting information; Hassan et al. Nitric oxide homeostasis
Figure S3. Inhibition of NO reduction by high NO concentrations.
Panel A shows the results for preliminary experiments similar to that shown in Fig. 4, but with higher final doses of NO. The log-log plot shows the cell-specific rates of NO reduction ( 𝑣
NO fmol cell
-1
h
-1
) against the NO concentrations in the liquid ( [NO] aq
) for single time increments during depletion of four consecutive doses (7, 55, 292, and 3000 ppmv ; 1 ppmv NO in headspace gives 2.12 nM in the liquid when in equilibrium). 𝑣
NO
during depletion of the first three doses shows reasonably similar concentration dependency, while the cells that had been exposed to the final high dose of NO
( 3000 ppmv ; [NO] aq
≈ 6
µM
) had clearly lower rates by a factor of 2–3. In these experiments, OD was measured throughout the experiments, and the cell numbers were found to increase in proportion with the cumulated NO reduction throughout the depletion of the first three doses, but not after the fourth high dose. Thus, growth was evidently permanently impeded by exposure to such high NO concentrations. Panel B shows the result for one of the three replicate vials used in the final experiment for determining K 𝑚NO
and 𝑣 𝑚𝑎𝑥NO
. In this case, the last high dose ( 881 ppmv ;
[NO] aq
≈ 1.87
µM
if in equilibrium) apparently resulted in a more transient depression in NO reduction.
4

Supporting information; Hassan et al. Nitric oxide homeostasis
Figure S4. Estimated cell-specific rates of NO-reduction ( 𝒗
𝐍𝐎
, fmol cell -1 h -1 ) for individual time increments.
The rates are plotted against the estimated concentration of NO in the aqua
( [NO] aq
). Panel A shows the entire range, and Panel B shows the results for [NO]
LP
< 300 nM . All data shown are for NO depletion through the first 2–3 injections of NO < 500 ppmv (= [NO] aq
<
1
µM
). The estimated 𝑣
NO
for [NO] aq
> 300 nM is more variable than at lower concentrations.
5

Supporting information; Hassan et al. Nitric oxide homeostasis
Figure S5. The relationship between the estimated NO concentration at the cell surface
( [𝐍𝐎] 𝐜𝐞𝐥𝐥
) and that in the bulk liquid ( [𝐍𝐎] 𝐚𝐪
).
The panel shows the ratio
[NO]
[NO] cell
plotted against aq
[NO] aq
(log scale), together with the theoretical curve for spheres with radius 0.4
µm
, 𝑣 𝑚𝑎𝑥NO
=
3.56 fmol cell
-1
h
-1
, and K 𝑚NO
= 34 nM . There is good agreement for [NO] aq
> 20 nM , but for lower concentrations, there is much experimental noise. A negative ratio means that the estimated
[NO] cell
is negative.
6